Methods and compositions using stearoyl-CoA desaturase to identify triglyceride reducing therapeutic agents

ABSTRACT

The use of screening assays based on the role of human stearoyl-CoA desaturase-1 (“hSCD1”) in human diseases, disorders or conditions relating to serum levels of triglyceride, VLDL, HDL, LDL, total cholesterol, or production of secretions from mucous membranes, monounsaturated fatty acids, wax esters, and the like, is disclosed. Also disclosed are conventions useful in the prevention and/or treatment of such diseases.

[0001] This application claims priority of U.S. Provisional ApplicationNo. 60/184,526, filed Feb. 24, 2000, U.S. Provisional Application No.60/221,697, filed Jul. 31, 2000, and U.S. Provisional Application No.60/255,771, filed Dec. 15, 2000, the disclosures of which are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field ofstearoyl-CoA desaturase and its involvement in various human diseases.Stearoyl-CoA desaturase, and the gene encoding it, are useful foridentification and development of therapeutic agents for the treatmentof such diseases.

BACKGROUND OF THE INVENTION

[0003] Acyl desaturase enzymes catalyze the formation of double bonds infatty acids derived from either dietary sources or de novo synthesis inthe liver. Mammals synthesize four desaturases of differing chain lengthspecificity that catalyze the addition of double bonds at the Δ9, Δ6, Δ5and Δ4 positions. Stearoyl-CoA desaturases (SCDs) introduce a doublebond in the Δ9-position of saturated fatty acids. The preferredsubstrates are palmitoyl-CoA (16:0) and stearoyl-CoA (18:0), which areconverted to palmitoleoyl-CoA (16:1) and oleoyl-CoA (18:1),respectively. The resulting mono-unsaturated fatty acids are substratesfor incorporation into phospholipids, triglycerides, and cholesterolesters.

[0004] A number of mammalian SCD genes have been cloned. For example,two genes have been cloned from rat (SCD1, SCD2) and four SCD genes havebeen isolated from mouse (SCD1, 2, 3, and 4). A single SCD gene, SCD1,has been characterized in humans.

[0005] While the basic biochemical role of SCD has been known in ratsand mice since the 1970's (Jeffcoat R. and James, A T. 1984. ElsevierScience, 4: 85-112; de Antueno, R J. 1993. Lipids 28(4)285-290), it hasnot, prior to this invention, been directly implicated in human diseaseprocesses. Studies in non-human animals have obscured our understandingof the role of SCD in humans due to the well documented differences inthe biochemical processes in different species. In rodents, for example,lipid and cholesterol metabolism is particularly obscured by the absenceof Cholesterol Ester Transport Protein (CETP) (see Foger, B. et al.1999. J. Biol. Chem. 274(52) 36912).

[0006] Further, the existence of multiple SCD genes in mice and ratsadds additional complexity to determining the specific role of each ofthese genes in disease processes. Differences in tissue expressionprofiles, substrate specificity, gene regulation and enzyme stabilitymay be important in elucidating which SCD gene plays the dominant rolein each disorder. Most previous SCD studies assess SCD gene function bymeasuring mRNA levels or by measuring levels of monounsaturated fattyacids as an indirect measure of SCD enzyme activity. In both these casesthis analysis may be misleading. In the latter method it has beenparticularly misleading and difficult to discern the relativecontribution of SCD1 to the plasma desaturation index (the ratio ofmonounsaturated fatty acids to saturated fatty acids of a specific chainlength) due to the fact that multiple SCD enzymes may contribute to theproduction of monounsaturated fatty acids. Prior to this invention, therelative contributions of the multiple SCD isoforms to the desaturationindex was unknown. In summary, previous studies have not differentiatedwhich SCD isoforms play a major role in the total desaturase activity asmeasured by the desaturation index.

[0007] Recent work in in vitro chicken hepatocyte cell culture relatesdelta-9 desaturase activity to impaired triacylglycerol secretion(Legrand, P. and Hermier, D. (1992) Int. J. Obesity 16, 289-294;Legrand, P., Mallard, J., Bernard-Griffiths, M. A., Douaire, M., andLemarchal, P. Comp. Biochem. Physiol. 87B, 789-792; Legrand, P.,Catheline, D., Fichot, M.-C., Lemarchal, P. (1997) J. Nutr. 127,249-256). This work did not distinguish between isoforms of delta-9desaturase that may exist in the chicken, once again failing to directlyimplicate a specific SCD enzyme to account for a particular biologicaleffect, in this case, impaired triglyceride secretion.

[0008] Nor does this in vitro work correlate well to humans becausesubstantial differences exist between chicken and human lipoproteinmetabolism in vivo. Such differences include the presence, in chicken,of entirely different lipoproteins, such as vitellogenin, and distinctprocesses such as the massive induction of hepatic triglyceridesynthesis during ovulation. Other differences such as the type oflipoproteins used for cholesterol transport and the process of secretionof dietary triglyceride in chylomicrons are well documented. These majordifferences between avians and mammals mean that extrapolation from theavians to mammals in the area of triglyceride metabolism must beconsidered provisional pending confirmation in humans.

[0009] Two other areas of background art form an important basis to theinstant invention. Firstly, this invention relates to cholesterol andlipid metabolism, which in humans has been intensely studied. Sincecholesterol is highly apolar, it is transported through the bloodstreamin the form of lipoproteins consisting essentially of a core of apolarmolecules such as cholesterol ester and triglyceride surrounded by anenvelope of amphipathic lipids, primarily phospholipids. In humans,approximately 66% of cholesterol is transported on low densitylipoprotein (LDL) particles, about 20% on high density lipoprotein (HDL)particles, and the remainder on very low density lipoprotein (VLDL)particles. An excellent reference to the basic biochemistry ofcholesterol metabolism in humans and other organisms is found at Biologyof Cholesterol. Ed. Yeagle, P. CRC Press, Boca Raton, Fla., 1988.

[0010] Secondly, this invention takes advantage of new findings from theAsebia mouse (Gates, et al. (1965) Science. 148:1471-3). This mouse is anaturally occurring genetic variant mouse that has a well known defectin sebaceous glands, resulting in hair loss and scaly skin. The Asebiamouse has recently been reported to have a deletion in SCD1 resulting inthe formation of an early termination site in exon 3 of the SCD1 gene.Animals homozygous for this mutation, or a distinct deletion allelewhich encompasses exons 1-4, do not express detectable amounts of thewild-type SCD1 mRNA transcript (November 1999. Nature Genetics. 23:268et seq.; and PCT patent publication WO 00/09754)]. Since the full extentof this naturally occurring deletion is unknown, it is also unknown ifother genes neighboring SCD1, or elsewhere in the genome, could also beinvolved in the Asebia phenotype. In order to specifically study theactivity of SCD1 in these disease processes, a specific SCD1 knockoutmouse is required. The prior work on this variant has focused on therole of this mutation in skin disorders and not on triglyceride or VLDLmetabolism.

[0011] It is an object of the instant invention to identify diseases anddisorders that are linked specifically to SCD1 biological activity inhumans, and in a preferred embodiment, diseases and disorders oftriglyceride metabolism. It is a further object to develop screeningassays to identify and develop drugs to treat those diseases, disordersand related conditions. Further, it is an object of this invention toprovide compositions for use in treating these disease, disorders andrelated conditions.

BRIEF SUMMARY OF THE INVENTION

[0012] This invention discloses, for the first time, the role of humanstearoyl-CoA desaturase-1 (“hSCD1”) in a wide range of human diseasesand disorders. In particular, SCD1 biological activity in humans isdirectly related to serum levels of triglycerides and VLDL. In addition,SCD1 biological activity also affects serum levels of HDL, LDL, and/ortotal cholesterol, reverse cholesterol transport, and the production ofsecretions from mucous membranes, monounsaturated fatty acids, waxesters, and/or the like.

[0013] It is an object of the present invention to provide a process orscreening assay for identifying, from a library of test compounds, atherapeutic agent which modulates the biological activity of said humanstearoyl-CoA desaturase (hSCD1) and is useful in treating a humandisorder or condition relating to serum levels of triglyceride or VLDL.Preferably, the screening assay identifies inhibitors of hSCD1 whichlower serum triglyceride levels and provide an importantcardioprotective benefit for humans.

[0014] It is also an object of the present invention to provide aprocess or screening assay for identifying, from a library of testcompounds, a therapeutic agent which modulates the biological activityof said human stearoyl-CoA desaturase (hSCD1) and is useful in treatinga human disorder or condition relating to serum levels of HDL, LDL,and/or total cholesterol, reverse cholesterol transport or theproduction of secretions from mucous membranes, monounsaturated fattyacids, wax esters, and/or the like

[0015] In one aspect, the present invention relates to vectorscomprising human stearoyl-CoA desaturase (hSCD1) genes and promotersequences and to recombinant eukaryotic cells, and cell lines,preferably mammalian cells, and most preferably human cells, and celllines, transfected so as to comprise such vectors and/or saidpolynucleotides and wherein said cells express hSCD1.

[0016] Disclosed herein is the full length promoter sequence for hSCD1,SEQ ID. No. 1.

[0017] It is also an object of the present invention to provide agentscapable of modulating the activity and/or expression of humanstearoyl-CoA desaturase 1 (hSCD1) as disclosed herein, especially wheresaid modulating ability was first determined using an assay comprisinghSCD1 biological activity or a gene encoding hSCD1. Pharmaceuticalcompositions comprising such agents are specifically contemplated.

[0018] It is a still further object of the present invention to provideagents wherein said agent is useful in treating, preventing and/ordiagnosing a disease or condition relating to hSCD1 biological activity.

[0019] It is a yet further object of the present invention to provide aprocess for preventing or treating a disease or condition in a patientafflicted therewith comprising administering to said patient atherapeutically or prophylactically effective amount of a composition asdisclosed herein.

[0020] In a pharmacogenomic application of this invention, an assay isprovided for identifying cSNPs (coding region single nucleotidepolymorphisms) in hSCD1 of an individual which are associated with humandisease processes or response to medication.

[0021] In other aspects, the present invention also provides a processfor diagnosing a disease or condition in a patient, commonly a humanbeing, suspected of being afflicted therewith, or at risk of becomingafflicted therewith, comprising obtaining a tissue sample from saidpatient and determining the level of activity of hSCD1 in the cells ofsaid tissue sample and comparing said activity to that of an equalamount of the corresponding tissue from a patient not suspected of beingafflicted with, or at risk of becoming afflicted with, said disease orcondition.

[0022] In other aspects, the present invention also provides a processfor diagnosing a disease or condition in a patient, commonly a humanbeing, suspected of being afflicted therewith, or at risk of becomingafflicted therewith, comprising obtaining a tissue sample from saidpatient and identifying mutations in the hSCD1 gene in the cells of saidtissue sample and comparing said gene to that of a corresponding tissuefrom a patient not suspected of being afflicted with, or at risk ofbecoming afflicted with, said disease or condition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1. Generation of SCD1 null mice (A) Targeting strategy forSCD1. A partial map of the genomic locus surrounding the Scd1 locus isshown. Homologous recombination resulted in the replacement of exons 1-6by neo 7 gene. Gene-targeting events were verified by Southern blotanalysis using EcoRI and probe A or B or by PCR analysis. (B) PCRanalysis demonstrating SCD −/− mice. In breeding heterozygotes,wild-type, heterozygotes and homozygotes were born in Mendelian fashion(+/+:+/−:−/−=21:43:20 x²=0.395). (C) Northern blot analysis. 20 μg oftotal RNA was isolated from the liver and subjected to Northern blotanalyses. Blots were probed with a mouse SCD1 and 2 cDNA fragments. (D)Immunoblot analysis of liver showed the absence of immunoreactive SCD inSCD1 −/− mice, whereas SCD1 protein was detected in liver tissue fromboth wild-type and heterozygote mice in a manner dependent on genedosage. (E) Liver SCD activity was abolished in SCD −/− mice. Asmentioned above, heterozygotes present an intermediate phenotype whencompared to wild-type and null littermates. Enzyme activity isrepresented as nanomoles of substrate desaturated per milligram ofprotein per minute. Data are denoted as the mean±SD (n=3).

[0024]FIG. 2. Plasma lipoprotein profiles in SCD1 Knock-out and AsebiaMale Mice. The top two panels depict the triglyceride content of thelipoprotein fractions, the bottom two panels depict the cholesterolcontent of the lipoprotein fractions.

[0025]FIG. 3. VLDL-triglyceride levels in Asebia (SCD1−/−) and SCD1+/−mice. Plasma lipoproteins were separated by fast performance liquidchromatography and the distribution of triglycerides among lipoproteinsin the various density fractions of the mice (n=3) were measured. SCD−/−(open circles), SCD1+/− (filled circles). The lipoprotein peaks forVLDL, LDL and HDL are indicated.

[0026]FIG. 4. Ratio of monounsaturated to saturated fatty acid in mouseplasma (the desaturation index) decreases in a manner directlyproportional to the level of SCD activity I. Comparison of SCD1knock-out and asebia mice to their respective controls.

[0027]FIG. 5 shows a linear regression analysis using a human data set.The ratio of 18:1/18:0 showed a significant relationship to TG levels(r²=0.39, p<0.0001) (Panel A), as well as a significant correlations toHDL levels (r²=0.12, p=0.0006) (Panel B). The experimental details arefurther described in Example 2.

[0028]FIG. 6 shows a linear regression analysis indicating a weakrelationship between the relative level of 16:1/16:0 to plasma TG levelswas observed (r²=0.05, p=0.03). Experimental details are in Example 2.

[0029]FIG. 7 shows a linear regression analysis of those individualswith a high HDL phenotype (>90^(th) percentile). These individualsdemonstrated a significant relationship between the 18:1/18:0 ratio andTG levels (r²=0.40, p<0.005).

[0030]FIG. 8 shows an observed relationship between 18:1/18:0 and TGlevels was observed in a family (HA-1) that segregates a high HDLphenotype. Using linear regression analysis, a significant relationshipbetween 18:1/18:0 and TG was observed (r²=0.36, p=0.005 (Panel A)).Panel B shows a significant relationship between 18:1/18:0 ratio and HDLlevels in this family (r²=0.32, p=0.009).

[0031]FIG. 9 shows the relationship observed between the 18:1/18:0 ratioand TG levels (r²=0.49, p=0.0009) when only persons with low HDL(<5^(th) percentile) are considered.

[0032]FIG. 10 shows an analysis of a family (NL-001), which segregated alow HDL phenotype of unknown genetic etiology and tended towards therelationships observed in FIGS. 5-9.

[0033]FIG. 11 shows an analysis of family NL-0020 which segregated anABCA1 mutation and tended towards the relationships noted in FIGS. 5-9.

[0034]FIG. 12 is a plasma fatty acid analysis showing the relationshipbetween the 18:1/18:0 ratio and TG levels (r2=0.56, p=0.02) (Panel A),HDL levels (r2=0.64, p=0.0095) (Panel B) and total cholesterol levels(r2=0.50, p=0.03) in nine persons with Familial Combined Hyperlipidemia(FCHL) (Panel C).

[0035]FIG. 13 is a plasma fatty acid analysis showing a significantlyelevated 18:1/18:0 ratio in hyperlipidemic mice (HcB-19) when comparedto unaffected controls of the parental strain (C3H).

[0036]FIG. 14. Location of regulatory sequences and binding sites inhomologous region of the mouse SCD1 and human SCD1 promoter and5′-flanking regions. The top scale denotes the position relative to thetranscriptional start site. Important promoter sequence elements areindicated.

[0037]FIG. 15. Human HepG2 cells cultured and treated with a range ofdoses of arachidonic acid, DHA or 10 μg/ml cholesterol or EPA asindicated. Total mRNA was isolated and quantified.

[0038]FIG. 16. TLC of lipid extracts from skin (A and B) and eyelids (Cand D) of wild-type, heterozygotes and SCD −/− mice. Total lipids wereextracted from eyelids of wild-type, heterozygotes and SCD −/− mice.Lipid extracts were pooled and analyzed by high performance TLC (HPTLC,A and C; hexane ether/ether/acetic acid=90:25:1, B and D; Benzene:hexane; 65:35). Same amounts of lipid extract (from 0.5 mg of eyelid)were subjected in each lane. Each lane represents lipids from eyelids oftwo mice.

[0039]FIG. 17 shows an assay for SCD1 desaturase activity by quantifyingtransfer of ³H from stearate to water. The figure shows a time course of³H-water production at room temperature. Microsomes from wild typelivers were used for this experiment. A turnover number for SCD1activity under these conditions was estimated at 2 mmol/min/mg protein,which is about half that observed at 37° C.

[0040]FIG. 18 shows validation that the assay monitors specificallySCD1-dependent desaturation of stearoyl-CoA. Livers were collected fromwild type and SCD1 knockout mice following 3 days on a high carbohydratefat-free diet (this induces SCD1 expression in liver by about 50-fold),homogenized and a portion used for microsome purification. ³H-waterproduction was determined for 15 minutes at RT (room temperature) inboth homogenate and microsome preparations at equivalent proteinconcentration, followed by quenching the reaction with acid and usingcharcoal to separate substrate from product. Quenching the sample withacid prior to the addition of substrate offers a blank, or controlcontaining background radioactivity without any desaturation reaction.The figure shows that desaturation activity in microsomes is greatlyenriched compared to the homogenate for wild type livers, while themicrosomes from the −/− SCD1 knockout animal has very little activity.The “window”, or SCD1-dependent desaturation in this assay, is a highlyvisible and significant 160-fold difference between wild type and SCD1knockout microsomes.

[0041]FIG. 19 shows inhibition of SCD1 with 3 known fatty acidinhibitors. Microsomes from wild type mice were used to test theeffectiveness of three known inhibitors of SCD1: conjugated linoleicacid (CLA), 9-thia stearic acid (9-thia) and sterculic acid (SA). PanelA shows that when added as the free fatty acid none were effective tosuppress SCD1 activity. However, panel B shows that if pre-conjugated toCoA (done by incubating the microsomes with CoA and ATP prior to theaddition of 3H-stearoyl CoA) the three inhibitors show graded inhibitionof SCD1 with sterculic acid suppressing nearly 100% of the activity forthe preincubation condition. This experiment establishes that SCD1activity can be inhibited with known inhibitors but they appear torequire conjugation with CoA. An important use of this screening assayis to find small molecules that are potent inhibitors of SCD1 biologicalactivity without conjugation to CoA.

[0042]FIG. 20(A) Demonstration that stearoyl-CoA mass limits thekinetics and magnitude of the 3H-production signal we are taking as ameasure of SCD1-dependent desaturation. This experiment is essentially arepeat of that shown in FIG. 17 with the exception that at 30 mins anadditional aliquot of stearoyl-CoA mass/radioactivity was added,resulting in the second exponential production of 3H signal. This showsthat the amount of stearyol-CoA limits the reaction as expected forSCD1-catalyzed desaturation. (B) Demonstration that the experiment isadaptable to high throughput. All previous experiments were done wherethe total reaction volume was 1.1 ml (0.2 ml reaction buffer containingmicrosomes, 0.2 ml 6% PCA to quench the reaction and 0.7 ml 10% charcoalsolution to sediment the unreacted substrate). The experimentillustrated in B was done with a total reaction volume of 0.31 ml (0.1ml reaction buffer with microsomes, 0.01 ml 60% PCA to quench and 0.2 ml10% charcoal to sediment).

DEFINITIONS

[0043] “Isolated” in the context of the present invention with respectto polypeptides or polynucleotides means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polynucleotideor polypeptide present in a living organism is not isolated, but thesame polynucleotide or polypeptide, separated from some or all of theco-existing materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0044] The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form.” As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform.

[0045] The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, claimed polypeptide which has a purity of preferably0.001%, or at least 0.01% or 0.1%; and even desirably 1% by weight orgreater is expressly contemplated.

[0046] The term “coding region” refers to that portion of a gene whicheither naturally or normally codes for the expression product of thatgene in its natural genomic environment, i.e., the region coding in vivofor the native expression product of the gene. The coding region can befrom a normal, mutated or altered gene, or can even be from a DNAsequence, or gene, wholly synthesized in the laboratory using methodswell known to those of skill in the art of DNA synthesis.

[0047] In accordance with the present invention, the term “nucleotidesequence” refers to a heteropolymer of deoxyribonucleotides (for DNA) orribonucleotides (for RNA). Generally, DNA segments encoding the proteinsprovided by this invention are assembled from cDNA fragments and shortoligonucleotide linkers, or from a series of oligonucleotides, toprovide a synthetic gene which is capable of being expressed in arecombinant transcriptional unit comprising regulatory elements derivedfrom a microbial or viral operon.

[0048] The term “expression product” means that polypeptide or proteinthat is the natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

[0049] The term “fragment,” when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

[0050] The term “primer” means a short nucleic acid sequence that ispaired with one strand of DNA and provides a free 3′OH end at which aDNA polymerase starts synthesis of a deoxyribonucleotide chain.

[0051] The term “promoter” means a region of DNA involved in binding ofRNA polymerase to initiate transcription, and includes all nucleotidesupstream (5′) of the transcription start site.

[0052] As used herein, reference to a DNA sequence includes both singlestranded and double stranded DNA. Thus, the specific sequence, unlessthe context indicates otherwise, refers to the single strand DNA of suchsequence, the duplex of such sequence with its complement (doublestranded DNA) and the complement of such sequence.

[0053] The present invention further relates to a polypeptide which hasthe deduced amino acid sequence, as well as fragments, analogs andderivatives of such polypeptide.

[0054] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide, means a polypeptide which retains essentially the samebiological function or activity as such polypeptide. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide. Suchfragments, derivatives and analogs must have sufficient similarity tothe SCD1 polypeptide so that activity of the native polypeptide isretained.

[0055] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide, and ispreferably a recombinant polypeptide.

[0056] The fragment, derivative or analog of the SCD1 polypeptide may be(i) one in which one or more of the amino acid residues are substitutedwith a conserved or non-conserved amino acid residue (preferably aconserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code, or (ii) one in whichone or more of the amino acid residues includes a substituent group, or(iii) one in which the mature polypeptide is fused with anothercompound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol), or (iv) one in which theadditional amino acids are fused to the mature polypeptide, such as aleader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

[0057] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0058] In accordance with the foregoing, the present invention alsorelates to an isolated stearoyl-CoA desaturase encoded by the isolatedpolynucleotide of the invention.

[0059] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0060] As used herein, the terms “portion,” “segment,” and “fragment,”when used in relation to polypeptides, refer to a continuous sequence ofresidues, such as amino acid residues, which sequence forms a subset ofa larger sequence. For example, if a polypeptide were subjected totreatment with any of the common endopeptidases, such as trypsin orchymotrypsin, the oligopeptides resulting from such treatment wouldrepresent portions, segments or fragments of the starting polypeptide.When used in relation to polynucleotides, such terms refer to theproducts produced by treatment of said polynucleotides with any of thecommon endonucleases.

DETAILED SUMMARY OF THE INVENTION

[0061] The present invention relates to the activity of humanstearoyl-CoA desaturase-1 in human disease processes. In accordancetherewith, compounds that specifically modulate human stearoyl-CoAdesaturase-1 activity or expression level are useful in the treatment ofa human disorder or condition relating to serum levels of triglycerideor VLDL, and provide an important cardioprotective benefit whenadministered to humans. Compounds that modulate hSCD1 activity orexpression are also useful for modulating serum levels of HDL, LDL,and/or total cholesterol, and/or reverse cholesterol transport. Finally,compounds that modulate hSCD1 activity or expression are also useful formodulating the production of secretions from mucous membranes,monounsaturated fatty acids, wax esters, and the like.

[0062] The SCD1 Gene and Protein

[0063] Human Stearoyl-CoA Desaturase-1 (also called SCD1, hSCD andhSCD1) has been identified with the full cDNA sequence first released toGenBank as GenBank Accession Y13647 (also NM005063) dated Jun. 6, 1997.Further descriptions of SCD1, including partial promoter sequences canbe found on GenBank under the following accession numbers:gb|AF097514.1|AF097514; dbj|AB032261.1|AB032261; gb|AF116616.1|AF116616;ref|XM_(—)005719.1|; gb|AF113690.1|AF113690; and gb|S70284.1|S70284

[0064] In one aspect the present invention relates to uses of anisolated polynucleotide comprising a non-genomic polynucleotide havingat least 90% identity, preferably 95% identity, most preferably at leasta 98% identity to the sequence of human stearoyl-CoA reductase-1,especially where said sequences are the same and including any of thecomplements of any of the foregoing.

[0065] The full promoter sequence of hSCD1 is SEQ ID. No. 1 and FIG. 14illustrates the functional elements conserved between the mouse andhuman SCD1 promoter regions.

[0066] In one aspect the present invention relates to uses of anisolated polypeptide having at least 90% identity, preferably 95%identity, most preferably at least a 98% identity to human stearoyl-CoAreductase-1, especially where said sequences are the same. Thepolypeptide sequence has been previously disclosed and can be found atthe following SwissProtein database accession series: ACCESSION No.000767; PID g3023241; VERSION 000767 GI:3023241; DBSOURCE: swissprot:locus ACOD_HUMAN, accession 000767. Alternatively, the polypeptidesequence can be determined from the cDNA sequence references providedabove.

[0067] SCD1 in Human Disease Processes

[0068] As disclosed herein, a number of human diseases and disorders arethe result of aberrant SCD1 biological activity and may be amelioratedby modulation of SCD1 biological activity using therapeutic agents.

[0069] The most significant teaching of the present disclosure relatesto the role of SCD1 in modulating serum triglyceride and VLDL levels inhumans. Two major findings are established herein. Firstly, Example 1below shows that the lipoprotein profiles of SCD1 knock-out micedemonstrate a 65% reduction in serum triglyceride and VLDL levels. Thesecorrespond with the lipoprotein profiles of Asebia mice which are alsoincluded herein. The lipoprotein profiles of both the Asebia and theSCD1 knock-out mouse were not previously known but due to the targetedand specific nature of the engineered mutation, the correlation of SCD1activity and serum triglyceride levels can be drawn with certainty.While other SCD isoforms may play a role in triglyceride levels, thisdata indicates that SCD1, specifically, plays the major role in thisprocess.

[0070] There are significant differences in lipoprotein metabolismbetween mouse and humans, and while the foregoing data are convincing inthe mouse for a major and specific role for SCD1 in modulation oftrigylceride levels, this still needed confirmatory experiments inhumans. The second major finding, therefore, presented in Example 2,below, demonstrates a significant correlation between SCD activity inhumans and levels of serum triglycerides. It has thus been discoveredthat SCD1 biological activity in humans is directly related to levels ofserum triglycerides.

[0071] In accordance with the present invention, the Asebia mousephenotype (first described by Gates, et al. (1965) Science. 148:1471-3)shows a major and significant alteration in serum lipoprotein profileincluding a large reduction in triglyceride and VLDL levels. Inaddition, these animals have a large decrease in liver content ofcholesterol esters. In accordance therewith, effective inhibition ofSCD1 activity would lead to a reduction in triglyceride levels, due todecreased availability of monounsaturated fatty acids. Monounsaturatedfatty acids are the preferred substrate for the enzyme responsible fortriglyceride (TG) synthesis from fatty acids and glycerol phosphate(viz., glycerol phosphate acyl transferase (GPAT)).

[0072] Also in accordance with the disclosure herein, increasedesterification of cholesterol prevents the toxic accumulation of freecholesterol in liver, and the increase in the availability ofcholesterol esters and triglycerides also facilitates their secretion inthe form of VLDL. Increased cholesterol esterification in macrophagesmay also enhance the formation of foam cells and thereby contribute toatherosclerotic lesion development. Thus, the inhibition of SCD activitymay have the added effect of reducing the level of VLDL particles in thebloodstream and inhibiting atherosclerosis.

[0073] Further in accordance with the present invention, inhibition ofSCD1 is also advantageous in increasing the formation of HDL atperipheral tissues. In a healthy individual, cellular cholesterol ispredominantly in the esterified form, with low levels of freecholesterol. Acyl-CoA:cholesterol acyltransferase (ACAT) is the enzymeresponsible for esterifying cholesterol using monounsaturated fattyacyl-CoA's as a preferred substrate. SCD generates the monounsaturatedproducts, which are then available for cholesterol esterification byACAT. The increased flux of free cholesterol out of cells and throughHDL is thought to be therapeutically beneficial because it would signifyenhanced “reverse cholesterol transport” (RCT).

[0074] Inhibition of SCD1 is also useful in increasing reversecholesterol transport (RCT) without necessarily raising the serum HDLlevel. Serum HDL level is a surrogate marker for the process of RCT,which in fact preferably is measured by the overall flux of cholesterolfrom peripheral tissues to the liver. The invention identifiesmodulators of SCD1 biological activity as effective therapeutic agentsfor increasing RCT. RCT can be directly measured, for example, byinjecting radiolabelled cholesterol ether incorporated into HDLparticles directly into the blood, and measuring the clearance rate (therate at which it is taken up into the liver of an organism).

[0075] In accordance with the present invention, it has been found thatmodulation of SCD1 activity in the liver and other tissues results in anincrease in SR-B1, a liver receptor which removes HDL from thecirculation, thus increasing RCT with less obvious effects on HDL levelsin the blood. The linkage between SCD1 biological activity and SR-B1mRNA expression has not previously been identified. Previous work hasestablished that SR-B1-overexpressing mice are cardioprotected,demonstrating reduced atherogenesis and reduced cardiovascular disease.This understanding also suggests for the first time that certaintherapeutic agents, such as inhibitors of SCD1 biological activity, mayincrease RCT without any obvious changes on HDL levels. This is achievedby obtaining a balanced increase in both HDL formation in peripheraltissues and HDL removal by the liver.

[0076] The experiment compared SR-B1 mRNA expression in the liver of +/+versus −/− SCD1 mice (strains as described in the Examples below). Whenexpressed relative to the +/+ animal on chow diet the results show thefollowing for changes in ABCA1 and SR-B1 mRNA levels: genotype diet ABC1SR-B1 +/+ Chow 1 1 −/− Chow 0.7 11 +/+ Hi Cholesterol 1.1 27 −/− HiCholesterol 0.4 27

[0077] The changes in ABC1 are not significant while those shown forSR-B1 on a chow diet are. An increase in SR-B1 expression indicatesincreased flux, or RCT, of cholesterol to the liver and may explain whythere is no observation of elevated HDL-C in the plasma of the −/− SCD1mouse. Increased RCT is further confirmed by the finding that −/−animals on high cholesterol diet have a gall bladder roughly 10-timesthe size of the +/+ animals and which are engorged with bile. Theseobservations are consistent with increased removal of cholesterol by theliver, hence increased RCT. Further, the apparently identical increasein SR-B1 in +/+ and −/− mice may not reflect an identical phenotype orbiological process in these animals.

[0078] Inhibition of SCD expression may also affect the fatty acidcomposition of membrane phospholipids, as well as triglycerides andcholesterol esters. The fatty acid composition of phospholipidsultimately determines membrane fluidity, while the effects on thecomposition of triglycerides and cholesterol esters can affectlipoprotein metabolism and adiposity.

[0079] The present invention also relates to the involvement of SCD1 inother human disorders or conditions relating to serum levels of HDL,LDL, and total cholesterol as well as the role of SCD1 in other humandisorders or conditions relating to the production of secretions frommucous membranes, monounsaturated fatty acids, wax esters, and the like.The invention encompasses modulators of SCD1 that are useful fortreating these disorders.

[0080] Previous work not using human subjects has shown that aberrantSCD biological activity in those organisms (but not specifying whichisoform of SCD was responsible) may be implicated in various skindiseases, as well as such diverse maladies as cancer and multiplesclerosis, non-insulin-dependent diabetes mellitus, hypertension,neurological diseases, skin diseases, eye diseases, immune disorders,and cancer. Modulators discovered using the processes of the presentinvention would thereby also find use in treating those diseases anddisorders in human subjects.

[0081] In Example 4, transcription regulating proteins for SCD1 areidentified. These proteins are targets for compounds that increase ordecrease SCD1 expression in cells, thereby influencing, eitherpositively or negatively, SCD1 biological activity of cells. PPAR-gammaand SREBP are examples. Compounds which are known to act through suchtranscription regulators may now be identified as relevant for treatingthe SCD1 related diseases and disorders now identified in humans.

[0082] Screening Assays

[0083] The present invention provides screening assays employing thehSCD1 gene and/or protein for use in identifying therapeutic agents foruse in treating a disorder or condition relating to serum levels oftriglyceride, VLDL, HDL, LDL, total cholesterol, reverse cholesteroltransport, the production of secretions from mucous membranes,monounsaturated fatty acids, wax esters, and the like.

[0084] “SCD1 Biological Activity”

[0085] “SCD1 biological activity” as used herein, especially relating toscreening assays, is interpreted broadly and contemplates all directlyor indirectly measurable and identifiable biological activities of theSCD1 gene and protein. Relating to the purified SCD1 protein, SCD1biological activity includes, but is not limited to, all thosebiological processes, interactions, binding behavior, binding-activityrelationships, pKa, pD, enzyme kinetics, stability, and functionalassessments of the protein. Relating to SCD1 biological activity in cellfractions, reconstituted cell fractions or whole cells, these activitiesinclude, but are not limited the rate at which the SCD introduces acis-double bond in its substrates palmitoyl-CoA (16:0) and stearoyl-CoA(18:0), which are converted to palmitoleoyl-CoA (16:1) and oleoyl-CoA(18:1), respectively, and all measurable consequences of this effect,such as triglyceride, cholesterol, or other lipid synthesis, membranecomposition and behavior, cell growth, development or behavior and otherdirect or indirect effects of SCD1 activity. Relating to SCD1 genes andtranscription, SCD1 biological activity includes the rate, scale orscope of transcription of genomic DNA to generate RNA; the effect ofregulatory proteins on such transcription, the effect of modulators ofsuch regulatory proteins on such transcription; plus the stability andbehavior of mRNA transcripts, post-transcription processing, mRNAamounts and turnover, and all measurements of translation of the mRNAinto polypeptide sequences. Relating to SCD1 biological activity inorganisms, this includes but is not limited biological activities whichare identified by their absence or deficiency in disease processes ordisorders caused by aberrant SCD1 biological activity in thoseorganisms. Broadly speaking, SCD1 biological activity can be determinedby all these and other means for analyzing biological properties ofproteins and genes that are known in the art.

[0086] The screening assays contemplated by the present invention mayalso employ isoforms of SCD from humans or other organisms thatdemonstrate similar biological activity as hSCD1 so long as they succeedin identifying therapeutic agents for human diseases. The functionalequivalency of delta-9 desaturases from vertebrates has been recognizedby those in the art. Consequently, specific embodiments of the presentinvention may employ one or more functionally equivalent delta-9desaturase enzymes from another vertebrate species to identifytherapeutic agents useful for humans. Functionally equivalentdesaturases include all of the mouse, rat, cow, pig or chicken SCDsidentified above, in addition to the genes identified at the UniGeneCluster Hs.119597 SCD for Stearoyl-CoA desaturase (delta-9-desaturase).See also LocusLink: 6319; OMIM: 604031 or HomoloGene: Hs. 119597. Otherknown delta-9 desaturases include pig: O02858 (swiss-prot); and cow:AF188710 (NCBI, [6651449, Genbank])

[0087] Selected Model Organism Protein Similarities

[0088] (organism, protein reference and percent identity and length ofaligned amino acid (aa) region) H. sapiens: SP:O00767- 100%/358 aa M.musculus: PIR:A32115- 83%/357 aa R. norvegicus: SP:P07308- 84%/357 aa D.melanogaster: PID:g1621653- 57%/301 aa C. elegans: PID:g3881877- 52%/284aa S. cerevisiae: PID:e243949- 36%/291 aa B. Taurus O02858 85%/359 aa S.Scrofa 6651449 86%/334 aa

[0089] Design and Development of SCD Screening Assays

[0090] The present disclosure facilitates the development of screeningassays that may be cell based, cell extract (i.e. microsomal assays),cell free (i.e. transcriptional) assays, and assays of substantiallypurified protein activity. Such assays are typically radioactivity orfluorescence based (i.e. fluorescence polarization or fluorescenceresonance energy transfer or FRET), or they may measure cell behavior(viability, growth, activity, shape, membrane fluidity, temperaturesensitivity etc). Alternatively, screening may employ multicellularorganisms, including genetically modified organisms such as knock-out orknock-in mice, or naturally occurring genetic variants. Screening assaysmay be manual or low throughput assays, or they may be high throughputscreens which are mechanically/robotically enhanced.

[0091] The aforementioned processes afford the basis for screeningprocesses, including high throughput screening processes, fordetermining the efficacy of potential therapeutic and diagnostic drugsfor treating the diseases described herein, preferably diseases in whichincreased or decreased activity or expression of stearoyl-CoA desaturase(hSCD1 of the invention) plays a key role in mediating such disease.

[0092] As such this invention relates to a method for identifying, suchas from a library of test compounds, a therapeutic agent which is usefulin humans for the treatment of a disorder or condition relating to serumlevels of triglyceride, VLDL, HDL, LDL, total cholesterol or productionof secretions from mucous membranes, monounsaturated fatty acids, waxesters, and the like, comprising

[0093] a) providing a screening assay having SCD1 biological activity;

[0094] b) contacting said screening assay with a test compound; and

[0095] c) subsequently measuring said biological activity;

[0096] wherein a test compound which modulates said biological activityis said therapeutic agent, or an analog thereof.

[0097] In one aspect, the present invention relates to a process foridentifying, from a library of test compounds, a therapeutic agent whichis useful in humans for the treatment of a disorder or conditionrelating to serum levels of triglyceride or very low density lipoprotein(VLDL) comprising

[0098] a) providing a screening assay having stearoyl-Coenzyme Adesaturase type 1 (SCD1) biological activity as a component thereof;

[0099] b) contacting said SCD1 activity with a test compound;

[0100] c) administering to a human a compound found to modulate saidactivity in (b); and

[0101] (d) detecting a change in serum level of triglyceride or VLDL insaid human following said administering;

[0102] thereby identifying an agent useful in the treatment of adisorder or condition relating to serum levels of triglyceride or verylow density lipoprotein (VLDL).

[0103] In one embodiment, said agent is an antagonist or inhibitor ofSCD1 biological activity. In another specific embodiment thereof, saidagent is an agonist of SCD1 biological activity.

[0104] In another embodiment, where said modulator is an inhibitor, saidinhibitor does not substantially inhibit the biological activity in ahuman of a delta-5 desaturase, delta-6 desaturase or fatty acidsynthetase

[0105] In one embodiment of the present invention, the assay processfurther comprises the step of assaying said therapeutic agent to furtherselect compounds which do not substantially inhibit in a human theactivity of delta-5 desaturase, delta-6 desaturase or fatty acidsynthetase.

[0106] In specific embodiments, the present invention also encompasses aprocess wherein said SCD1 biological activity is measured by an assayselected from among:

[0107] a) SCD1 polypeptide binding affinity;

[0108] b) SCD1 desaturase activity in microsomes;

[0109] c) SCD1 desaturase activity in a whole cell assay

[0110] d) quantification of SCD1 gene expression level; and

[0111] e) quantification of SCD1 protein level.

[0112] Specific embodiments of such an assay may employ a recombinantcell as disclosed herein.

[0113] The present invention also relates to a process wherein theidentified compound is further selected from among those compounds thatdo not substantially inhibit in humans the biological activity ofdelta-5 desaturase, delta-6 desaturase or fatty acid synthetase.

[0114] In other specific embodiments, the present invention contemplatesemploying SCD1 nucleic acid as disclosed herein and/or SCD1 polypeptideas disclosed herein for use in identifying compounds useful fortreatment of a disorder or condition relating to serum levels oftriglyceride or VLDL.

[0115] The assays disclosed herein essentially require the measurement,directly or indirectly, of an SCD1 biological activity. Those skilled inthe art can develop such assays based on well known models, and manypotential assays exist. For measuring whole cell activity of SCD1directly, methods that can be used to quantitatively measure SCDactivity include for example, measuring thin layer chromatographs of SCDreaction products over time. This method and other methods suitable formeasuring SCD activity are well known (Henderson Henderson R J, et al.1992. Lipid Analysis: A Practical Approach. Hamilton S. Eds. New Yorkand Tokyo, Oxford University Press. pp 65-111.) Gas chromatography isalso useful to distinguish mononunsaturates from saturates, for exampleoleate (18:1) and stearate (18:0) can be distinguished using thismethod. A description of this method is in the examples below. Thesetechniques can be used to determine if a test compound has influencedthe biological activity of SCD1, or the rate at which the SCD introducesa cis-double bond in its substrate palmitate (16:0) or stearate (18:0)to produce palmitolyeoyl-CoA (16:1) or oleyoyl-CoA (18:1), respectively.

[0116] In a preferred embodiment, the invention employs a microsomalassay having a measurable SCD1 biological activity. A suitable assay maybe taken by modifying and scaling up the rat liver microsomal assayessentially as described by Shimomura et al. (Shimomura, I., Shimano,H., Korn, B. S., Bashmakov, Y., and Horton, J. D. (1998). Tissues arehomogenized in 10 vol. of buffer A (0.1M potassium buffer, pH 7.4). Themicrosomal membrane fractions (100,000×g pellet) are isolated bysequential centrifugation. Reactions are performed at 37° C. for 5 minwith 100 μg of protein homogenate and 60 μM of [1-¹⁴C]-stearoyl-CoA(60,000 dpm), 2 mM of NADH, 0.1M of Tris/HCl buffer (pH 7.2). After thereaction, fatty acids are extracted and then methylated with 10% aceticchloride/methanol. Saturated fatty acid and monounsaturated fatty acidmethyl esters are separated by 10% AgNO₃-impregnated TLC usinghexane/diethyl ether (9:1) as developing solution. The plates aresprayed with 0.2% 2′, 7′-dichlorofluorescein in 95% ethanol and thelipids are identified under UV light. The fractions are scraped off theplate, and the radioactivity is measured using a liquid scintillationcounter.

[0117] Specific embodiments of such SCD1 biological activity assay takeadvantage of the fact that the SCD reaction produces, in addition to themonounsaturated fatty acyl-CoA product, H₂O. If ³H is introduced intothe C-9 and C-10 positions of the fatty-acyl-CoA substrate, then some ofthe radioactive protons from this reaction will end up in water. Thus,the measurement of the activity would involve the measurement ofradioactive water. In order to separate the labeled water from thestearate, investigators may employ substrates such as charcoal,hydrophobic beads, or just plain old-fashioned solvents in acid pH.

[0118] In a preferred embodiment, screening assays measure SCD1biological activity indirectly. Standard high-throughput screeningassays centre on ligand-receptor assays. These may be fluorescence basedor luminescence based or radiolabel detection. Enzyme immunoassays canbe run on a wide variety of formats for identifying compounds thatinteract with SCD1 proteins. These assays may employ prompt fluorescenceor time-resolved fluorescence immunoassays which are well known. P³²labeled ATP, is typically used for protein kinase assays. Phosphorylatedproducts may be separated for counting by a variety of methods.Scintillation proximity assay technology is an enhanced method ofradiolabel assay. All these types of assays are particularly appropriatefor assays of compounds that interact with purified or semi-purifiedSCD1 protein.

[0119] In a preferred embodiment, the assay makes use of 3H-stearoyl CoA(with the 3H on the 9 and 10 carbon atoms), the substrate for SCD1.Desaturation by SCD1, produces oleoyl CoA and 3H-water molecules. Thereaction is run at room temperature, quenched with acid and thenactivated charcoal is used to separate unreacted substrate from theradioactive water product. The charcoal is sedimented and amount ofradioactivity in the supernatant is determined by liquid scintillationcounting. This assay is specific for SCD1-dependent desaturation asjudged by the difference seen when comparing the activity in wild typeand SCD1-knockout tissues. Further, the method is easily adapted to highthroughput as it is cell-free, conducted at room temperature and isrelatively brief (1 hour reaction time period versus previous period of2 days). This procedure is illustrated more fully in FIGS. 17 to 20.

[0120] While the instant disclosure sets forth an effective workingembodiment of the invention, those skilled in the art are able tooptimize the assay in a variety of ways, all of which are encompassed bythe invention. For example, charcoal is very efficient (>98%) atremoving the unused portion of the stearoyl-CoA but has the disadvantageof being messy and under some conditions difficult to pipette. It maynot be necessary to use charcoal if the stearoyl-CoA complexsufficiently aggregates when acidified and spun under moderate g-force.This can be tested by measuring the signal/noise ratio with and withoutcharcoal following a desaturation reaction. There are also otherreagents that would efficiently sediment stearoyl-CoA to separate itfrom the 3H-water product.

[0121] As shown in FIG. 20 (Panel A) the amount of stearoyl-CoA limitsthe kinetics and magnitude of the ³H-DPM signal monitored asSCD1-dependent desaturation activity. However, not all of thestearoyl-CoA was consumed by SCD1; >90% remains unavailable to SCD1either because other enzymes present in the microsomes (e.g., acyltransferase reactions) utilize it as a substrate and compete with SCD1and/or stearoyl-CoA is unstable under the conditions of the experiment.These possibilities may be examined by monitoring incorporation of thelabel into phospholipids or by including a buffer mixture (Mg++, ATP andCoA) that would regenerate stearoyl-CoA from stearate and CoA.

[0122] As shown in FIG. 20 (Panel B) the assay can be done in a smallvolume appropriate for high throughput screening. A preferred embodimentwould employ a microcentrifuge satisfactory for spinning 96 well plates.

[0123] The following assays are also suitable for measuring SCD1biological activity in the presence of potential therapeutic agents.These assays are also valuable as secondary screens to further selectSCD1 specific modulators, inhibitors or agonists from a library ofpotential therapeutic agents.

[0124] Cellular based desaturation assays can also be used. By trackingthe conversion of stearate to oleate in cells (3T3L1 adipocytes areknown to have high SCD1 expression and readily take up stearate whencomplexed to BSA) we can evaluate compounds found to be inhibitory inthe primary screen for additional qualities or characteristics such aswhether they are cell permeable, lethal to cells, and/or competent toinhibit SCD1 activity in cells. This cellular based assay may employ arecombinant cell line containing a delta-9 desaturase, preferably hSCD1(human SCD1). The recombiant gene is optionally under control of aninducible promoter and the cell line preferably over-expresses SCD1protein.

[0125] Other assays for tracking other SCD isoforms could be developed.For example, rat and mouse SCD2 is expressed in brain. In a preferredembodiment, a microsome preparation is made from the brain as previouslydone for SCD1 from liver. The object may be to find compounds that wouldbe specific to SCD1. This screen would compare the inhibitory effect ofthe compound for SCD1 versus SCD2.

[0126] Although unlikely, it is possible that a compound “hit” in theSCD1 assay may result from stimulation of an enzyme present in themicrosome preparation that competitively utilizes stearoyl-CoA at theexpense of that available for SCD1-dependent desaturation. This wouldmistakenly be interpreted as SCD1 inhibition. One possibility to examinethis problem would be incorporation into phospholipids of theunsaturated lipid (stearate). By determining effects of the compounds onstimulation of stearate incorporation into lipids researchers are ableto evaluate this possibility.

[0127] Cell based assays may be preferred, for they leave the SCD1 genein its native format. Particularly promising for SCD1 analysis in thesetypes of assays are fluorescence polarization assays. The extent towhich light remains polarized depends on the degree to which the tag hasrotated in the time interval between excitation and emission. Since themeasurement is sensitive to the tumbling rate of molecules, it can beused to measure changes in membrane fluidity characteristics that areinduced by SCD1 activity—namely the delta-9 desaturation activity of thecell. An alternate assay for SCD1 involves a FRET assay. FRET assaysmeasure fluorescence resonance energy transfer which occurs between afluorescent molecule donor and an acceptor, or quencher. Such an assaymay be suitable to measure changes in membrane fluidity or temperaturesensitivity characteristics induced by SCD1 biological activity.

[0128] The screening assays of the invention may be conducted using highthroughput robotic systems. In the future, preferred assays may includechip devices developed by, among others, Caliper, Inc., ACLARABioSciences, Cellomics, Inc., Aurora Biosciences Inc., and others.

[0129] In other embodiments of the present invention, SCD1 biologicalactivity can also be measured through a cholesterol efflux assay thatmeasures the ability of cells to transfer cholesterol to anextracellular acceptor molecule and is dependent on ABCA1 function. Astandard cholesterol efflux assay is set out in Marcil et al.,Arterioscler. Thromb. Vasc. Biol. 19:159-169, 1999, incorporated byreference herein for all purposes.

[0130] Preferred assays are readily adapted to the format used for drugscreening, which may consist of a multi-well (e.g., 96-well, 384 well or1536 well or greater) format. Modification of the assay to optimize itfor drug screening would include scaling down and streamlining theprocedure, modifying the labeling method, altering the incubation time,and changing the method of calculating SCD1 biological activity etc. Inall these cases, the SCD1 biological activity assay remains conceptuallythe same, though experimental modifications may be made.

[0131] Another preferred cell based assay is a cell viability assay forthe isolation of SCD1 inhibitors. Overexpression of SCD decreases cellviability. This phenotype can be exploited to identify inhibitorycompounds. This cytotoxicity may be due to alteration of the fatty acidcomposition of the plasma membrane. In a preferred embodiment, the humanSCD1 cDNA would be placed under the control of an inducible promoter,such as the Tet-On Tet-Off inducible gene expression system (Clontech).This system involves making a double stable cell line. The firsttransfection introduces a regulator plasmid and the second wouldintroduce the inducible SCD expression construct. The chromosomalintegration of both constructs into the host genome would be favored byplacing the transfected cells under selective pressure in the presenceof the appropriate antibiotic. Once the double stable cell line wasestablished, SCD1 expression would be induced using the tetracycline ora tetracycline derivative (eg. Doxycycline). Once SCD1 expression hadbeen induced, the cells would be exposed to a library of chemicalcompounds for HTS of potential inhibitors. After a defined time period,cell viability would then be measured by means of a fluorescent dye orother approach (e.g. turbidity of the tissue culture media). Those cellsexposed to compounds that act to inhibit SCD1 activity would showincreased viability, above background survival. Thus, such an assaywould be a positive selection for inhibitors of SCD1 activity based oninducible SCD1 expression and measurement of cell viability.

[0132] An alternative approach is to interfere with the desaturasesystem. The desaturase system has three major proteins: cytochrome b₅,NADH (P)-cytochrome b₅ reductase, and terminal cyanide-sensitivedesaturase. Terminal cyanide-sensitive desaturase is the product of theSCD gene. SCD activity depends upon the formation of a stable complexbetween the three aforementioned components. Thus, any agent thatinterferes with the formation of this complex or any agent thatinterferes with the proper function of any of the three components ofthe complex would effectively inhibit SCD activity.

[0133] Another type of modulator of SCD1 activity involves a 33 aminoacid destabilization domain located at the amino terminal end of thepre-SCD1 protein (Mziaut et al., PNAS 2000, 97: p 8883-8888). It ispossible that this domain may be cleaved from the SCD1 protein by an asyet unknown protease. This putative proteolytic activity would thereforeact to increase the stability and half-life of SCD1. Inhibition of theputative protease, on the other hand, would cause a decrease in thestability and half life of SCD1. Compounds which block or modulateremoval of the destabilization domain therefore will lead to reductionsin SCD1 protein levels in a cell. Therefore, in certain embodiments ofthe invention, a screening assay will employ a measure of proteaseactivity to identify modulators of SCD1 protease activity. The firststep is to identify the specific protease which is responsible forcleavage of SCD1. This protease can then be integrated into a screeningassay. Classical protease assays often rely on splicing a proteasecleavage site (i.e., a peptide containing the cleavable sequencepertaining to the protease in question) to a protein, which isdeactivated upon cleavage. A tetracycline efflux protein may be used forthis purpose. A chimera containing the inserted sequence is expressed inE. coli. When the protein is cleaved, tetracycline resistance is lost tothe bacterium. In vitro assays have been developed in which a peptidecontaining an appropriate cleavage site is immobilized at one end on asolid phase. The other end is labeled with a radioisotope, fluorophore,or other tag. Enzyme-mediated loss of signal from the solid phaseparallels protease activity. These techniques can be used both toidentify the protease responsible for generating the mature SCD1protein, and also for identifying modulators of this protease for use indecreasing SCD1 levels in a cell.

[0134] In another aspect, the present invention relates to a process fordetermining the ability of an agent to modulate the activity of a humanstearoyl-CoA desaturase, comprising the steps of:

[0135] (a) contacting the agent under suitable conditions with the humanstearoyl-CoA desaturase of the invention at a predetermined level ofsaid agent;

[0136] (b) determining if the activity of said stearoyl-CoA desaturasechanges after said contact,

[0137] thereby determining if said agent has modulated said activity.

[0138] Such an assay may be carried out as a cell free assay employing acellular fractional, such as a microsomal fraction, obtained byconventional methods of differential cellular fractionation, mostcommonly by ultracentrifugation methods. In specific embodiments, suchmodulation may be an increase or decrease in the activity of thedesaturase.

[0139] These results suggest that inhibitors of SCD biological activity,such as hSCD1, in a human, may have the beneficial effect of reducingtriglycerides and/or increasing HDL levels. In addition, increased SCDactivity is also associated with increased body weight index. Thisresult identifies hSCD1 as a useful target for identifying agents formodulating obesity and related conditions. In these human data results,SCD biological activity was measured via the surrogate marker of theratio of 18:1 to 18:0 fatty acids in the total plasma lipid fraction.This marker indirectly measures hSCD1 biological activity.

[0140] In a further aspect, the present invention relates to a processfor determining the ability of an agent to modulate the activity of ahuman stearoyl-CoA desaturase in cells expressing the human stearoyl-CoAdesaturase of the invention, comprising the steps of:

[0141] (a) contacting the agent under suitable conditions with aeukaryotic cell expressing the human stearoyl-CoA desaturase of theinvention at a predetermined level of said agent and under conditionswhere said agent may or may not modulate the expression level of saiddesaturase;

[0142] (b) determining if the activity of said stearoyl-CoA desaturasechanges after said contact,

[0143] thereby determining if said agent has modulated said expressionlevel.

[0144] In specific embodiments of said processes, the modulation may bean increase or decrease in activity of the desaturase and cells usefulin these processes are preferably mammalian cells, most preferably humancells, and include any of the recombinant cells disclosed herein.

[0145] SCD1 Recombinant Cell Lines

[0146] In certain embodiments, the present invention contemplates use ofa SCD1 gene or protein in a recombinant cell line. SCD1 recombinant celllines may be generated using techniques known in the art, and those morespecifically set out below.

[0147] The present invention also relates to vectors which containpolynucleotides of the present invention, and host cells which aregenetically engineered with vectors of the invention, especially wheresuch cells result in a cell line that can be used for assay of hSCD1activity, and production of SCD1 polypeptides by recombinant techniques.

[0148] Host cells are preferably eukaryotic cells, preferably insectcells of Spodoptera species, most especially SF9 cells. Host cells aregenetically engineered (transduced or transformed or transfected) withthe vectors, especially baculovirus) of this invention which may be, forexample, a cloning vector or an expression vector. Such vectors caninclude plasmids, viruses and the like. The engineered host cells arecultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transformants or amplifying the genes ofthe present invention. The culture conditions, such as temperature, pHand the like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

[0149] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0150] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0151] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0152] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0153] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein. Such transformation will be permanent and thus giverise to a cell line that can be used for further testing. Such celllines used for testing will commonly be mammalian cells, especiallyhuman cells.

[0154] As representative examples of appropriate hosts, there may bementioned Spodoptera Sf9 (and other insect expression systems) andanimal cells such as CHO, COS or Bowes melanoma; adenoviruses; plantcells, and even bacterial cells, etc, all of which are capable ofexpressing the polynucleotides disclosed herein. The selection of anappropriate host is deemed to be within the knowledge of those skilledin the art based on the teachings herein. For use in the assay methodsdisclosed herein, mammalian, especially human, cells are preferred.

[0155] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, especially where the Baculovirus/SF9vector/expression system is used, into which a sequence of the inventionhas been inserted, in a forward or reverse orientation. In a preferredaspect of this embodiment, the construct further comprises regulatorysequences, including, for example, a promoter, operably linked to thesequence. Large numbers of suitable vectors and promoters are known tothose of skill in the art, and are commercially available. The followingvectors are provided by way of example; Bacterial: pQE70, pQE60, pQE-9(Qiagen), pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A,pNH16a, pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any otherplasmid or vector may be used as long as they are replicable and viablein the host.

[0156] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lac, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-1. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0157] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)). A preferredembodiment utilizes expression from insect cells, especially SF9 cellsfrom Spodoptera frugiperda.

[0158] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0159] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), Wu et al, Methods in Gene Biotechnology (CRCPress, New York, N.Y., 1997), Recombinant Gene Expression Protocols, inMethods in Molecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa,N.J., 1997), and Current Protocols in Molecular Biology, (Ausabel et al,Eds.,), John Wiley & Sons, NY (1994-1999), the disclosures of which arehereby incorporated by reference in their entirety.

[0160] Transcription of the DNA encoding the polypeptides of the presentinvention by eukaryotic cells, especially mammalian cells, mostespecially human cells, is increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, a cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

[0161] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTrp1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal or C-terminal identificationpeptide imparting desired characteristics, e.g., stabilization orsimplified purification of expressed recombinant product.

[0162] Use of a Baculovirus-based expression system is a preferred andconvenient method of forming the recombinants disclosed herein.Baculoviruses represent a large family of DNA viruses that infect mostlyinsects. The prototype is the nuclear polyhedrosis virus (AcMNPV) fromAutographa californica, which infects a number of lepidopteran species.One advantage of the baculovirus system is that recombinantbaculoviruses can be produced in vivo. Following co-transfection withtransfer plasmid, most progeny tend to be wild type and a good deal ofthe subsequent processing involves screening. To help identify plaques,special systems are available that utilize deletion mutants. By way ofnon-limiting example, a recombinant ACMNPV derivative (called BacPAK6)has been reported in the literature that includes target sites for therestriction nuclease Bsu36I upstream of the polyhedrin gene (and withinORF 1629) that encodes a capsid gene (essential for virus viability).Bsf36I does not cut elsewhere in the genome and digestion of the BacPAK6deletes a portion of the ORF1629, thereby rendering the virusnon-viable. Thus, with a protocol involving a system like Bsu36I-cutBacPAK6 DNA most of the progeny are non-viable so that the only progenyobtained after co-transfection of transfer plasmid and digested BacPAK6is the recombinant because the transfer plasmid, containing theexogenous DNA, is inserted at the Bsu36I site thereby rendering therecombinants resistant to the enzyme. [see Kitts and Possee, A methodfor producing baculovirus expression vectors at high frequency,BioTechniques, 14, 810-817 (1993). For general procedures, see King andPossee, The Baculovirus Expression System: A Laboratory Guide, Chapmanand Hall, New York (1992) and Recombinant Gene Expression Protocols, inMethods in Molecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa,N.J., 1997), at Chapter 19, pp. 235-246.

[0163] In accordance with the foregoing, the present invention furtherrelates to vectors comprising a polynucleotide of the invention and torecombinant eukaryotic cells expressing the stearoyl-CoA desaturase ofthe present invention, preferably wherein said cell is a mammalian cell,most preferably a human cell.

[0164] The present invention further relates to processes for using thepolynucleotides, enzymes, and cells disclosed herein in a process fordetermining the ability of an agent to modulate the expression of saidhuman stearoyl-CoA desaturase in cells expressing said humanstearoyl-CoA desaturase of the invention, comprising the steps of:

[0165] (a) contacting the agent under suitable conditions with aeukaryotic cell expressing the human stearoyl-CoA desaturase of theinvention at a predetermined level of said agent;

[0166] (b) determining if the expression level of said stearoyl-CoAdesaturase changes after said contact,

[0167] thereby determining if said agent has modulated said expressionlevel.

[0168] Alternatively, the screening assay may employ a vector constructcomprising the hSCD1 promoter sequence of SEQ ID. No. 3 operably linkedto a reporter gene. Such a vector can be used to study the effect ofpotential transcription regulatory proteins, and the effect of compoundsthat modulate the effect of those regulatory proteins, on thetranscription of SCD1. An example of this type of vector is the pSCD-500plasmid described in the examples below. Reporter gene constructs suchas this are commonly used to indicate whether a test compound hassucceeded in activating the transcription of genes under the control ofa particular promoter.

[0169] In specific embodiments, the present invention contemplates aprocess wherein said modulation is an increase or decrease in saidexpression level and where said cell may be a mammalian cell, especiallya human cell, including any of the recombinant cells disclosed herein.In one embodiment, the expression level is determined by determining thelevel of messenger RNA produced after contact of said cell with saidagent.

[0170] Factors that may modulate gene expression include transcriptionfactors such as, but not limited to, retinoid X receptors (RXRs),peroxisomal proliferation-activated receptor (PPAR) transcriptionfactors, the steroid response element binding proteins (SREBP-1 andSREBP-2), REV-ERBα, ADD-1, EBPα, CREB binding protein, P300, HNF 4, RAR,LXR, and RORα, NF-Y, C/EBPalpha, PUFA-RE and related proteins andtranscription regulators. Screening assays designed to assess thecapacity of test compounds to modulate the ability of thesetranscription factors to transcribe SCD1 are also contemplated by thisinvention.

[0171] Physiological benefits of an increase or decrease in the activityor expression of hSCD1 include, but are not limited to, decreased plasmatriglycerides and/or increased plasma HDL leading to cardioprotectivebenefit, therapeutic benefit in Type II diabetes, weight loss, improvedgland secretions, and decreased chance of malignancy. Thus, thedetermination of the ability of agents to modulate such activity orexpression affords an opportunity to discover useful therapeutic agentsproducing such effects.

[0172] In addition, variations in hSCD1 gene expression, function,stability, catalytic activity and other characteristics may be due toallelic variations in the polynucleotide sequences encoding suchenzymes. The processes disclosed according to the present invention maylikewise be used to determine such genomic effects on expression ofhSCD1. Using the processes of the present invention, such variations maybe determined at the level of DNA polymorphism within the hSCD1 geneand/or promoter sequences. Such effects lead to the elucidation ofassociations between such polymorphisms and predisposition to cancer,neurological disease, skin disease, obesity, diabetes, immune functionand lipid metabolism through both population and family-based geneticanalysis.

[0173] Finally, those skilled in the art are able to confirm therelevance of hSCD1 to human health by analogy to animal models. Wellknown animal disease models may be used to ascertain the effect of anhSCD1 modulator on the growth, development, or disease process in theseanimals. Additionally, models include genetically modified multicellularanimals, such as knock-out or knock-in mice (as detailed in the examplesbelow).

[0174] In a general aspect, the present invention relates to a processfor identifying a SCD1-modulating agent, comprising:

[0175] a) contacting under physiological conditions a chemical agent anda molecule having or inducing SCD1 activity;

[0176] b) detecting a change in the activity of said molecule having orinducing SCD1 activity following said contacting;

[0177] thereby identifying an SCD1 modulating agent.

[0178] In specific embodiments of the invention, said molecule having orinducing SCD1 activity is a polypeptide having such activity, or apolynucleotide encoding a polypeptide having such activity, or apolypeptide modulating the activity of a polynucleotide encoding apolypeptide having such activity.

[0179] In specific embodiments, said change in activity is an increasein activity or is a decrease in activity.

[0180] In addition, said contacting may be accomplished in vivo. In onesuch embodiment, said contacting in step (a) is accomplished byadministering said chemical agent to an animal afflicted with atriglyceride (TG)- or very low density lipoprotein (VLDL)-relateddisorder and subsequently detecting a change in plasma triglyceridelevel in said animal thereby identifying a therapeutic agent useful intreating a triglyceride (TG)- or very low density lipoprotein(VLDL)-related disorder. In such embodiment, the animal may be a human,such as a human patient afflicted with such a disorder and in need oftreatment of said disorder.

[0181] In specific embodiments of such in vivo processes, said change inSCD1 activity in said animal is a decrease in activity, preferablywherein said SCD1 modulating agent does not substantially inhibit thebiological activity of a delta-5 desaturase, delta-6 desaturase or fattyacid synthetase.

[0182] In such processes as just disclosed, the detected change in SCD1activity is detected by detecting a change in any, some or all of thefollowing:

[0183] a) SCD1 polypeptide binding affinity;

[0184] b) SCD1 desaturase activity in microsomes;

[0185] c) SCD1 desaturase activity in a whole cell;

[0186] d) SCD1 gene expression; or

[0187] e) SCD1 protein level.

[0188] In accordance with the foregoing, the present invention is alsodirected to a recombinant cell line comprising a recombinant SCD1protein as disclosed herein. In one such embodiment, the whole cell of(c) above is derived from such a cell line, preferably wherein said SCD1modulating agent does not substantially inhibit in humans the biologicalactivity of delta-5 desaturase, delta-6 desaturase or fatty acidsynthetase.

[0189] A recombinant cell line of the invention may also comprise theSCD1 promoter nucleic acid sequence of SEQ ID No. 1 operably linked to areporter gene construct. In a specific embodiment thereof, the wholecell of (c) above is derived from a recombinant cell of such a cellline.

[0190] In accordance with the disclosure herein, the present inventionis also directed to an isolated stearoyl-CoA desaturase encoded by thepolynucleotide sequence comprising SEQ ID No. [SCD1 cDNA] as well as areporter gene construct comprising the SCD1 promoter nucleic acidsequence of SEQ ID No. 1 operably linked to a reporter gene,advantageously including usable vectors comprising such the nucleicacids and constructs thereof. Likewise, the present invention alsocontemplates an isolated polypeptide having stearoyl-CoA reductaseactivity and a process as disclosed herein that successfully identifiesa chemical agent that binds to or interacts with such a polypeptide,which process comprises:

[0191] a) contacting such a polypeptide, or a cell expressing suchpolypeptide, with a chemical agent; and

[0192] b) detecting binding or interaction of the chemical agent withsaid polypeptide.

[0193] In specific embodiments of the process just described, thebinding of the chemical agent to the polypeptide is detected by a methodselected from the group consisting of:

[0194] a) direct detection of chemical agent/polypeptide binding;

[0195] b) detection of binding by competition binding assay; and

[0196] c) detection of binding by assay for SCD1 biological activity.

[0197] In such processes, modulation of the activity of such polypeptideis detected by a process comprising contacting the polypeptide or a cellexpressing the polypeptide with a compound that binds to the polypeptidein sufficient amount to modulate the activity of the polypeptide. Inspecific embodiments of this process, the molecule having or inducingSCD1 activity is selected from the group consisting of an SCD1 nucleicacid and/or SCD1 polypeptide as disclosed herein.

[0198] In accordance with the foregoing, following identification ofchemical agents having the desired modulating activity, the presentinvention also relates to a process for treating an animal, especially ahuman, such as a human patient, afflicted with a disease or conditionrelating to serum levels of triglyceride or VLDL comprising inhibitingSCD1 activity in said human. In a preferred embodiment, said inhibitionof SCD1 activity is not accompanied by substantial inhibition ofactivity of delta-5 desaturase, delta-6 desaturase or fatty acidsynthetase. In a specific embodiment, the present invention relates to aprocess for treating a human patient afflicted with a disorder orcondition relating to serum levels of triglyceride or VLDL comprisingadministering to said patient a therapeutically effective amount of anagent whose therapeutic activity was first identified by the process ofthe invention.

[0199] In accordance with the foregoing, the present invention alsorelates to a modulator of SCD1 activity and which is useful in humansfor treatment of a disorder or condition relating to serum levels oftriglyceride or VLDL wherein said activity was first identified by itsability to modulate SCD1 activity, especially where such modulation wasfirst detected using a process as disclosed herein according to thepresent invention. In a preferred embodiment thereof, such modulatingagent does not substantially inhibit fatty acid synthetase, delta-5desaturase or delta-6 desaturase of humans.

[0200] Thus, the present invention also relates to a process foridentifying a vertebrate delta-9 stearoyl-CoA desaturase-modulatingagent, comprising:

[0201] a) contacting under physiological conditions a chemical agent anda molecule having or inducing vertebrate delta-9 stearoyl-CoA desaturaseactivity;

[0202] b) detecting a change in the activity of said molecule having orinducing vertebrate delta-9 stearoyl-CoA desaturase activity followingsaid contacting;

[0203]  thereby identifying a vertebrate delta-9 stearoyl-CoA desaturasemodulating agent.

[0204] In a specific embodiment of such process, the contacting in step(a) is accomplished by administering said chemical agent to an animalafflicted with a disorder or condition related to serum levels oftriglyceride, VLDL, HDL, LDL, total cholesterol, reverse cholesteroltransport or production or secretion of mucous membranes,monounsaturated fatty acids, wax esters, and like parameters, detectinga change in the activity of said molecule having or inducing vertebratedelta-9 stearoyl-CoA desaturase activity following said contacting andthereby identifying a therapeutic agent useful in treating atriglyceride, VLDL, HDL, LDL, total cholesterol, reverse cholesteroltransport or production or secretion of mucous membranes,monounsaturated fatty acids, wax esters, and like disease-relateddisorder.

[0205] In accordance with the foregoing, the present invention furtherrelates to a process for treating a human patient afflicted with adisease or condition relating to serum levels of triglyceride, VLDL,HDL, LDL, total cholesterol, reverse cholesterol transport or productionor secretion of mucous membranes, monounsaturated fatty acids, waxesters, and like parameters, comprising administering to said humanpatient a therapeutically effective amount of an agent for which suchtherapeutic activity was identified by a process as disclosed hereinaccording to the invention.

[0206] In a preferred embodiments of such process, the modulating agentdoes not substantially inhibit fatty acid synthetase, delta-5 desaturaseor delta-6 desaturase of humans.

[0207] Test Compounds/Modulators/Library Sources

[0208] In accordance with the foregoing, the present invention alsorelates to therapeutic and/or diagnostic agents, regardless of molecularsize or weight, effective in treating and/or diagnosing and/orpreventing any of the diseases disclosed herein, preferably where suchagents have the ability to modulate activity and/or expression of thehSCD1 disclosed herein, and most preferably where said agents have beendetermined to have such activity through at least one of the screeningassays disclosed according to the present invention.

[0209] Test compounds are generally compiled into libraries of suchcompounds, and a key object of the screening assays of the invention isto select which compounds are relevant from libraries having hundreds ofthousands, or millions of compounds having unknown therapeutic efficacy.

[0210] Those skilled in the field of drug discovery and development willunderstand that the precise source of test extracts or compounds is notcritical to the screening procedure(s) of the invention. Accordingly,virtually any number of chemical extracts or compounds can be screenedusing the exemplary methods described herein. Examples of such extractsor compounds include, but are not limited to, plant-, fungal-,prokaryotic- or animal-based extracts, fermentation broths, andsynthetic compounds, as well as modification of existing compounds.Numerous methods are also available for generating random or directedsynthesis (e.g., semi-synthesis or total synthesis) of any number ofchemical compounds, including, but not limited to, saccharide-, lipid-,peptide-, and nucleic acid-based compounds. Synthetic compound librariesare commercially available from Brandon Associates (Merrimack, N.H.) andAldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant, and animal extractsare commercially available from a number of sources, including Biotics(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute(Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Inaddition, natural and synthetically produced libraries are produced, ifdesired, according to methods known in the art, e.g., by standardextraction and fractionation methods. Furthermore, if desired, anylibrary or compound is readily modified using standard chemical,physical, or biochemical methods.

[0211] Thus, in one aspect the present invention relates to agentscapable of modulating the activity and/or expression of humanstearoyl-CoA desaturase 1 (hSCD1) as disclosed herein, especially wheresaid modulating ability was first determined using an assay ofcomprising hSCD1 or a gene encoding hSCD1, or an assay which measureshSCD1 activity. As used herein the term “capable of modulating” refersto the characteristic of such an agent whereby said agent has the effectof changing the overall biological activity of hSCD1, either byincreasing or decreasing said activity, under suitable conditions oftemperature, pressure, pH and the like so as to facilitate suchmodulation to a point where it can be detected either qualitatively orquantitatively and wherein such modulation may occur in either an invitro or in vivo environment. In addition, while the term “modulation”is used herein to mean a change in activity, more specifically either anincrease or decrease in such activity, the term “activity” is not to belimited to specific enzymatic activity alone (for example, as measuredin units per milligram or some other suitable unit of specific activity)but includes other direct and indirect effects of the protein, includingincreases in enzyme activity due not to changes in specific enzymeactivity but due to changes (i.e., modulation) of expression ofpolynucleotides encoding and expressing said hSCD1 enzyme. Human SCD1activity may also be influenced by agents which bind specifically tosubstrates of hSCD1. Thus, the term “modulation” as used herein means achange in hSCD1 activity regardless of the molecular genetic level ofsaid modulation, be it an effect on the enzyme per se or an effect onthe genes encoding the enzyme or on the RNA, especially mRNA, involvedin expression of the genes encoding said enzyme. Thus, modulation bysuch agents can occur at the level of DNA, RNA or enzyme protein and canbe determined either in vivo or ex vivo.

[0212] In specific embodiments thereof, said assay is any of the assaysdisclosed herein according to the invention. In addition, the agent(s)contemplated by the present disclosure includes agents of any size orchemical character, either large or small molecules, including proteins,such as antibodies, nucleic acids, either RNA or DNA, and small chemicalstructures, such as small organic molecules.

[0213] In other aspects, the present invention contemplates agentswherein said agent is useful in treating, preventing and/or diagnosing adisease or condition which is identified as being SCD1 related accordingto this invention. Specific embodiments are directed to situationswherein the disease or condition includes, but is not limited to, serumlevels of triglyceride, VLDL, HDL, LDL, total cholesterol, reversecholesterol transport or production of secretions from mucous membranes,monounsaturated fatty acids, wax esters, and the like, cholesteroldisorders, lipidemias, cardiovascular disease, diabetes, obesity,baldness, skin diseases, cancer and multiple sclerosis, especially wherethe disease is a cardiovascular disease or a skin disease or where thecondition is baldness. In a preferred embodiment, such agents willincrease HDL levels in a patient and/or decrease triglyceride levels ina patient. Either or both effects are directly associated with reducedrisk of cardiovascular disease and coronary artery disease.

[0214] Some of the known modulators of SCD1 activity include conjugatedlinoleic acid especially trans-10, cis-12 isomer, and thiazoladinedionecompounds such as troglitazone.

[0215] While it is envisaged that any suitable mechanism for theinhibition or modulation of SCD1 activity can be used, three specificexamples of inhibitor classes are envisioned. One class includes thoseinhibitors that effectively inhibit SCD1 expression, such asthiazoladinedione compounds and polyunsaturated fatty acids. A secondclass includes those inhibitors that effectively inhibit SCD1 enzymaticactivity, such as thia-fatty acids, cyclopropenoid fatty acids, andcertain conjugated linoleic acid isomers. Finally, the third class ofinhibitors includes those agents that are capable of interfering withthe proteins essential to the desaturase system, such as those agentsthat interfere with cytochrome b₅, NADH (P)-cytochrome b₅ reductase, andterminal cyanide-sensitive desaturase.

[0216] For effectively inhibiting the expression of the SCD1 gene, it isenvisioned that any agent capable of disrupting the transcription of theSCD1 gene could be utilized. Since SCD1 is regulated by several knowntranscription factors (e.g. PPAR-γ, SREBP), any agent that affects theactivity of such transcription factors can be used to alter theexpression of the SCD1 gene. One group of such agents includesthiazoladine compounds which are known to activate PPAR-γ and inhibitSCD1 transcription. These compounds include Pioglitazone, Ciglitazone,Englitazone, Troglitazone, and BRL49653. Other inhibitory agents mayinclude polyunsaturated fatty acids, such as linoleic acid, arachidonicacid and dodecahexaenoic acid, which also inhibit SCD1 transcription.

[0217] For effectively inhibiting the enzymatic activity of the SCD1protein, it is envisaged that any agent capable of disrupting theactivity of the SCD1 protein could be utilized. For example, certainconjugated linoleic acid isomers are effective inhibitors of SCD1activity. Specifically, C is-12, trans-10 conjugated linoleic acid isknown to effectively inhibit SCD enzyme activity and reduce theabundance of SCD1 mRNA while C is-9, trans-11 conjugated linoleic aciddoes not. Cyclopropenoid fatty acids, such as those found in sterculaand cotton seeds, are also known to inhibit SCD activity. For example,sterculic acid (8-(2-octyl-cyclopropenyl)octanoic acid) and Malvalicacid (7-(2-octyl-cyclopropenyl)heptanoic acid) are C18 and C16derivatives of sterculoyl- and malvaloyl fatty acids, respectively,having cyclopropene rings at their Δ9 position. These agents inhibit SCDactivity by inhibiting Δ9 desaturation. Other agents include thia-fattyacids, such as 9-thiastearic acid (also called 8-nonylthiooctanoic acid)and other fatty acids with a sulfoxy moiety.

[0218] The known modulators of delta-9 desaturase activity are eithernot know to be useful for treating the diseases and disorders linked toSCD1 biological activity as claimed in this invention, or else they areotherwise unsatisfactory therapeutic agents. The thia-fatty acids,conjugated linoleic acids and cyclopropene fatty acids (malvalic acidand sterculic acid) are neither useful at reasonable physiologicaldoses, nor are they specific inhibitors of SCD1 biological activity,rather they demonstrate cross inhibition of other desaturases, inparticular the delta-5 and delta-6 desaturases by the cyclopropene fattyacids. These compounds may be useful for establishing control or testmodulators of the screening assays of the invention, but are not subjectto the claims of this invention. Preferred SCD1 modulators of theinvention have no significant or substantial impact on unrelated classesof proteins. In some cases, assays specific for the other proteins, suchas delta-5 and delta-6 activity, will also have to be tested to ensurethat the identified compounds of the invention do not demonstratesignificant or substantial cross inhibition.

[0219] The known non-specific inhibitors of SCD1 can be useful inrational design of a therapeutic agent suitable for inhibition of SCD1.All three inhibitors have various substitutions between carbons #9 and#10; additionally they require conjugation to Co-A to be effective; andare probably situated in a relatively hydrophobic active site. Thisinformation combined with the known X-ray co-ordinates for the activesite for plant (soluble) SCD can assist the “in silico” process ofrational drug design for therapeutically acceptable inhibitors specificfor SCD1.

[0220] This invention also provides an antibody which specifically bindsto human SCD1 having the amino acid sequence shown in the SwissProtaccession numbers listed above, and which thereby inhibits the activityof SCD1. The instant antibody can be a polyclonal antibody, a monoclonalantibody, or an SCD-binding fragment thereof. In one embodiment, theantibody is isolated, ie.e., an antibody free of any other antibodies.Methods of making and isolating antibodies are well known in the art(Harlow, et al. 1988. Antibodies: A Laboratory Manual; Cold SpringHarbor, N.Y., Cold Spring Harbor Laboratory).

[0221] This invention also provides an antisense oligonucleotide whichspecifically binds to human SCD1 mRNA, and which thereby reduces thelevel of SCD1 gene transcription. Methods of making and using antisensemolecules against known target genes are known in the art (Agarwal, S.(1996) Antisense Therapeutics. Totowa, N.J., Humana Press, Inc.)

[0222] Combinatorial and Medicinal Chemistry

[0223] Typically, a screening assay, such as a high throughput screeningassay, will identify several or even many compounds which modulate theactivity of the assay protein. The compound identified by the screeningassay may be further modified before it is used in humans as thetherapeutic agent. Typically, combinatorial chemistry is performed onthe modulator, to identify possible variants that have improvedabsorption, biodistribution, metabolism and/or excretion, or otherimportant therapeutic aspects. The essential invariant is that theimproved compounds share a particular active group or groups which arenecessary for the desired modulation of the target protein. Manycombinatorial chemistry and medicinal chemistry techniques are wellknown in the art. Each one adds or deletes one or more constituentmoieties of the compound to generate a modified analog, which analog isagain assayed to identify compounds of the invention. Thus, as used inthis invention, therapeutic compounds identified using an SCD1 screeningassay of the invention include actual compounds so identified, and anyanalogs or combinatorial modifications made to a compound which is soidentified which are useful for treatment of the disorders claimedherein.

[0224] Pharmaceutical Preparations and Dosages

[0225] In another aspect the present invention is directed tocompositions comprising the polynucleotides, polypeptides or otherchemical agents, including therapeutic, prophylactic or diagnosticagents, such as small organic molecules, disclosed herein according tothe present invention wherein said polynucleotides, polypeptides orother agents are suspended in a pharmacologically acceptable carrier,which carrier includes any pharmacologically acceptable diluent orexcipient. Pharmaceutically acceptable carriers include, but are notlimited to, liquids such as water, saline, glycerol and ethanol, and thelike, including carriers useful in forming sprays for nasal and otherrespiratory tract delivery or for delivery to the ophthalmic system. Athorough discussion of pharmaceutically acceptable carriers, diluents,and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES(Mack Pub. Co., N.J. current edition).

[0226] The inhibitors utilized above may be delivered to a subject usingany of the commonly used delivery systems known in the art, asappropriate for the inhibitor chosen. The preferred delivery systemsinclude intravenous injection or oral delivery, depending on the abilityof the selected inhibitor to be adsorbed in the digestive tract. Anyother delivery system appropriate for delivery of small molecules, suchas skin patches, may also be used as appropriate.

[0227] In another aspect the present invention further relates to aprocess for preventing or treating a disease or condition in a patientafflicted therewith comprising administering to said patient atherapeutically or prophylactically effective amount of a composition asdisclosed herein.

[0228] Diagnosis & Pharmacogenomics

[0229] In an additional aspect, the present invention also relates to aprocess for diagnosing a disease or condition in a patient, commonly ahuman being, suspected of being afflicted therewith, or at risk ofbecoming afflicted therewith, comprising obtaining a tissue sample fromsaid patient and determining the level of activity of hSCD1 in the cellsof said tissue sample and comparing said activity to that of an equalamount of the corresponding tissue from a patient not suspected of beingafflicted with, or at risk of becoming afflicted with, said disease orcondition. In specific embodiments thereof, said disease or conditionincludes, but is not limited to, cholesterol disorders, lipidemias,cardiovascular disease, diabetes, obesity, baldness, skin diseases,cancer and multiple sclerosis, especially wherein said disease is acardiovascular disease or a skin disease or said condition is baldness.

[0230] In an additional aspect, this invention teaches that hSCD1 haspharmacogenomic significance. Variants of hSCD1 including SNPs (singlenucleotide polymorphisms), cSNPs (SNPs in a cDNA coding region),polymorphisms and the like may have dramatic consequences on a subject'sresponse to administration of a prophylactic or therapeutic agent.Certain variants may be more or less responsive to certain agents. Inanother aspect, any or all therapeutic agents may have greater or lesserdeleterious side-effects depending on the hSCD1 variant present in thesubject.

[0231] In general, the invention discloses a process of selecting aprophylactic and/or therapeutic agent for administration to a subject inneed thereof comprising,

[0232] (a) determining at least a part of the hSCD1 nucleic acidsequence of said subject; and

[0233] (b) comparing said hSCD1 nucleic sequence to known variants ofhSCD1 nucleic acids;

[0234] wherein said known variants are correlated with responsiveness tosaid agent and said agent is selected for said subject on the basis of adesired correlation. In this method the correlation may be aprophylactic and/or therapeutic effect or it may be avoidance of adeleterious side effect, or any other desired correlation.

[0235] In a pharmacogenomic application of this invention, an assay isprovided for identifying cSNPs (coding region small nucleotidepolymorphisms) in hSCD1 of an individual which are correlated with humandisease processes or response to medication. The inventors haveidentified two putative cSNPs of hSCD1 to date:In exon 1, a C/A cSNP atnt 259, corresponding to a D/E amino acid change at position 8; and

[0236] in exon 5, a C/A cSNP at nt 905, corresponding to a L/M aminoacid change at position 224. (Sequence numbering according to GenBankAccession: AF097514). It is anticipated that these putative cSNPs may becorrelated with human disease processes or response to medication ofindividuals who contain those cSNPs versus a control population. Thoseskilled in the art are able to determine which disease processes andwhich responses to medication are so correlated.

[0237] In carrying out the procedures of the present invention it is ofcourse to be understood that reference to particular buffers, media,reagents, cells, culture conditions and the like are not intended to belimiting, but are to be read so as to include all related materials thatone of ordinary skill in the art would recognize as being of interest orvalue in the particular context in which that discussion is presented.For example, it is often possible to substitute one buffer system orculture medium for another and still achieve similar, if not identical,results. Those of skill in the art will have sufficient knowledge ofsuch systems and methodologies so as to be able, without undueexperimentation, to make such substitutions as will optimally servetheir purposes in using the methods and procedures disclosed herein.

[0238] In applying the disclosure, it should be kept clearly in mindthat other and different embodiments of the methods disclosed accordingto the present invention will no doubt suggest themselves to those ofskill in the relevant art.

EXAMPLE 1 Disruption of Stearoyl-CoA Desaturase 1 Gene in Mice CausesDecreased Plasma Triglycerides Levels, as Well as Other Defects in LipidMetabolism

[0239] This example identifies, for the first time, specific SCD1biological activities in mouse by characterizing an SCD1 gene specificknock-out mouse.

[0240] To investigate the physiological functions of SCD, we havegenerated SCD1 null (SCD1 −/−) mice. The lipoprotein profile of SCD1null (knock-out) mice demonstrates a striking decrease in triglyceride(i.e., VLDL) levels while maintaining approximately normal HDL and LDLlevels. This result confirms that a mutation in SCD1 is a causativemutation of a low triglyceride (TG) lipoprotein profile in mice, and isdistinct from other SCD isoforms in the mouse in this regard. Due to theseverity of this phenotype it is clear that other SCD isoforms areunlikely to affect TG levels to such a great extent.

[0241] Targeted Disruption of the SCD1 Gene

[0242]FIG. 1A shows the strategy used to knock out the SCD1 gene. Themouse SCD1 gene includes 6 exons. The first 6 exons of the gene werereplaced by a neomycin-resistant cassette by homologous recombination,resulting in the replacement of the complete coding region of the SCD1gene (FIG. 1A). The vector was electroporated into embryonic stem cellsand the clones that integrated the neo cassette were selected by growthon geneticin. Targeted ES clones were injected into C57BI/6 blastocystsyielding four lines of chimeric mice that transmitted the disruptedallele through the germ-line. The mutant mice were viable and fertileand bred with predicted Mendelian distributions. A PCR based screen toassay successful gene targeting of the SCD1 locus is shown in FIG. 1B.To determine whether the expression of the SCD1 gene was ablated weperformed Northern blot analysis (FIG. 1C) SCD1 mRNA is undetectable inliver of SCD1−/− mice and reduced by approximately 50% in SCD +/− mice.SCD2 mRNA was expressed at low levels in both SCD1−/− mice and wild-typemice. Consistent with Northern blot results, Western blot analysisshowed no immunoreactive SCD protein in liver from SCD −/− mice, whereasSCD1 protein was detectable in both heterozygous and wild-type livertissue in a manner dependent on gene dosage. SCD enzyme activity inliver, as measured by the rate of conversion of [1-¹⁴C stearoyl-CoA to[1-¹⁴C]oleate (FIG. 1E) was high in the wild-type mice but wasundetectable in the total extracts of livers of the SCD1−/− mice.

[0243] Lipid Analysis

[0244] Analysis of liver cholesterol ester (0.8±0.1 vs. 0.3±0.1 mg/gliver) and liver triglycerides (12.6±0.3 vs. 7.5±0.6 mg/g liver) showedthat SCD1 KO animals have lower amounts of both cholesterol esters andtriglycerides than wild-type controls. Plasma lipoprotein analysisshowed a decrease in plasma triglycerides (120.6±6.8 vs. 45.4±3.8) inSCD −/− mice compared to normal controls. These findings are similar tofindings in asebia mice. FIG. 2 records the plasma lipoprotein profileobtained using fast performance liquid chromatography. SCD1 Knock-Outmice showed a 65% reduction of triglyceride in VLDL fraction; but littleor no significant difference in LDL or HDL levels.

[0245] Asebia mice are compared with the SCD1 Knock-Out mice in FIG. 2.The findings are remarkably similar. Asebia mice plasma lipoproteinswere separated by fast performance liquid chromatography and thedistribution of triglycerides among lipoproteins in the various densityfractions of the mice (n=3) are shown. FIG. 3 shows an additionalexample of an Asebia mouse lipoprotein profile. These profiles showed amajor difference in the distribution of triglycerides in the VLDLfraction of the SCD−/− and SCD−/+ mice. The levels of triglycerides inthe SCD−/+ were 25 mg/dl in the VLDL, with very low levels in the LDLand HDL fractions. In contrast the SCD−/− had very low levels oftriglycerides in the three lipoprotein fractions.

[0246] Fatty Acid Analysis

[0247] We also determined the levels of monounsaturated fatty acids invarious tissues. Table 1 shows the fatty acid composition of severaltissues in wild-type and SCD −/− mice. The relative amounts ofpalmitoleate (16:1n-7) in liver and plasma from SCD −/− mice decreasedby 55% and 47% while those of oleate (18:1n-9) decreased by 35% and 32%,respectively. The relative amount of palmitoleate in white adiposetissue and skin of SCD −/− mice were decreased by more than 70%, whereasthe reduction of oleate in these tissues was less than 20% although thereduction was significant statistically. These changes in levels ofmonounsaturated fatty acids resulted in reduction of desaturationindices indicating reduction in desaturase activity. In contrast tothese tissues, the brain, which expresses predominantly the SCD2isoform, had a similar fatty acid composition and unaltered desaturationindex in both wild type and SCD−/− mice. We conclude that SCD1 plays amajor role in the production of monounsaturated fatty acids in theliver. TABLE I Fatty acid composition of several tissues from SCD1knockout mice Tissue total lipids from each mouse were extracted. Thelipids were methyl esterified and quantified by GLC as described underExperimental Procedures. Standard errors for all values were less than25% of the mean and were omitted from table for clarity. Bold valuesrepresent a statistical significance of p < 0.05 between wild-type andSCD −/− mice (student's t test). The values of monounsaturated/saturatedfatty acids were calculated from the mean value. 14:0 16:0 16:1n-7 18:018:1n-9 18:1n-7 18:2n-6 20:0 20:1n-9 Liver +/+ 0.8 25.9 1.1 16.1 16.21.6 16.3 0.0 0.0 −/− 1.0 27.2 0.5 22.8 10.6 1.0 13.9 0.0 0.0 Eyelid +/+1.3 15.0 2.4 9.3 19.6 3.4 5.2 5.2 24.1 −/− 1.9 22.4 1.5 20.3 16.1 3.76.8 7.5 3.7 WAT +/+ 3.3 27.6 5.2 5.7 35.1 1.9 19.5 0.0 0.0 −/− 2.7 29.21.5 14.8 29.1 1.7 18.7 0.0 0.0 Skin +/+ 3.5 29.3 4.0 9.7 32.4 2.1 15.00.0 0.0 −/− 3.1 30.7 1.4 14.2 28.1 1.8 17.6 0.0 0.0 Brain +/+ 1.1 25.70.8 21.6 16.5 3.1 1.1 0.0 0.0 −/− 1.1 26.2 0.8 21.1 15.8 3.3 1.2 0.0 0.0Eye Ball +/+ 2.9 31.3 1.6 28.5 19.1 2.6 0.0 0.0 0.0 −/− 3.2 32.3 1.529.8 18.8 2.4 0.0 0.0 0.0 20:1n-7 20:4n-6 22:6n-3 16:1/16:0 18:1n-9/18:018:1n-7/18:0 20:1n-9/20:C 20:1n-7/20:0 Liver +/+ 0.0 9.2 7.8 0.041 1.0060.100 0.000 0.000 −/− 0.0 6.8 8.8 0.018 0.465 0.044 0.000 0.000 Eyelid+/+ 9.6 0.9 0.8 0.160 2.108 0.366 4.635 1.846 −/− 7.4 1.7 1.5 0.0670.793 0.182 0.493 0.987 WAT +/+ 0.0 0.3 0.2 0.190 6.211 0.340 0.0000.000 −/− 0.0 0.4 0.8 0.050 1.967 0.115 0.000 0.000 Skin +/+ 0.0 0.9 0.70.136 3.351 0.219 0.000 0.000 −/− 0.0 0.9 0.8 0.045 1.982 0.128 0.0000.000 Brain +/+ 0.0 9.4 12.4 0.032 0.764 0.145 0.000 0.000 −/− 0.0 9.513.1 0.030 0.752 0.154 0.000 0.000 Eye Ball +/+ 0.0 2.9 7.2 0.051 0.6710.091 0.000 0.000 −/− 0.0 2.3 5.8 0.046 0.632 0.081 0.000 0.000

[0248]FIG. 4 (quantified in Table 2) demonstrate that SCD1 is a majorcontributor to the plasma desaturation indices (ratio of plasma18:1/18:0 or 16:1/16:0 in the total lipid fraction), as judged by plasmafatty acid analysis of both the SCD1 KO and asebia mice. In both animalmodels, a reduction of approximately 50% or greater is observed in theplasma desaturation indices. This demonstrates that the plasmadesaturation index is highly dependent on the function of SCD1 TABLE 2Fatty acid desaturation indices in asebia mutants and heterozygotesSex/genotype 18:1/18:0 16:1/16:0 Male +/− 1.393 0.044 Male −/− 0.7320.018 Female +/− 1.434 0.074 Female −/− 0.642 0.021 Female +/− 1.2030.081 Female −/− 0.574 0.022

[0249] Experimental Procedures for Knockout Mice:

[0250] Generation of the SCD1 Knockout Mice.

[0251] Mouse genomic DNA for the targeting vector was cloned from 129/SVgenomic library. The targeting vector construct was generated byinsertion of a 1.8-kb Xba I/Sac I fragment with 3′ homology as a shortarm and 4.4-kb Cla I/Hind III fragment with 5′ homology cloned adjacentto neo expression cassette. The construct also contains a HSV thymidinekinase cassette 3′ to the 1.8-kb homology arm, allowingpositive/negative selection. The targeting vector was linearized by NotI and electroporated into embryonic stem cells. Selection with geneticinand gancyclovior was performed. The clones resistant to both geneticinand gancyclovior were analyzed by Southern blot after EcoRI restrictionenzyme digestion and hybridized with a 0.4-kb probe located downstreamof the vector sequences. For PCR genotyping, genomic DNA was amplifiedwith primer A

[0252] 5′-GGGTGAGCATGGTGCTCAGTCCCT-3′ (SEQ ID NO: 2)

[0253] which is located in exon 6,primer B

[0254] 5′-ATAGCAGGCATGCTGGGGAT-3′ (SEQ ID NO: 3)

[0255] which is located in the neo gene (425 bp product, targetedallele), and primer C

[0256] 5′-CACACCATATCTGTCCCCGACAAATGTC-3′ (SEQ ID NO: 4)

[0257] which is located in downstream of the targeting gene (600 bpproduct, wild-type allele). PCR conditions were 35 cycles, each of 45sec at 94° C., 30 sec at 62° C., and 1 min at 72° C. The targeted cellswere microinjected into C57BI/6 blastocysts, and chimeric mice werecrossed with C57BL/6 or 129/SvEv Taconic females, and they gave thegerm-line transmission. Mice were maintained on a 12-h dark/light cycleand were fed a normal chow diet, a semi-purified diet or a dietcontaining 50% (% of total fatty acids) triolein, tripalmitolein ortrieicosenoin. The semi-purified diet was purchased from Harlan Teklad(Madison, Wis.) and contained: 20% vitamin free casein, 5% soybean oil,0.3% L-cystine, 13.2% Maltodextrin, 51.7% sucrose, 5% cellulose, 3.5%mineral mix (AIN-93G-MX), 1.0% vitamin mix (AIN-93-VX), 0.3% cholinebitartrate. The fatty acid composition of the experimental diets wasdetermined by gas liquid chromatography. The control diet contained 11%palmitic acid (16:0), 23% oleic acid (18:1n-9), 53% linoleic acid(18:2n-6) and 8% linolenic acid (18:3n-3). The high triolein dietcontained 7% 16:0, 50% 18:1n-9, 35% 18:2n-6 and 5% 18:3n-3.

[0258] Materials

[0259] Radioactive [-³²P]dCTP (3000 Ci/mmol) was obtained from DupontCorp. (Wilmington, Del.). Thin layer chromatography plates (TLC SilicaGel G60) were from Merck (Darmstadt, Germany). [1-¹⁴C]-stearoyl-CoA waspurchased from American Radiolabeled Chemicals, Inc. (St Louis, Mo.).Immobilon-P transfer membranes were from Millipore (Danvers, Mass.). ECLWestern blot detection kit was from Amersham-Pharmacia Biotech, Inc.(Piscataway, N.J.). All other chemicals were purchased from Sigma (StLouis, Mo.).

[0260] Lipid Analysis

[0261] Total lipids were extracted from liver and plasma according tothe method of Bligh and Dyer (Bligh and Dyer, 1959), and phospholipids,wax esters, free cholesterol, triglycerides and cholesterol esters wereseparated by silica gel high performance TLC. Petroleum hexane/diethylether/acetic acid (80:30:1) or benzene/hexane (65:35) was used as adeveloping solvent (Nicolaides and Santos, 1985). Spots were visualizedby 0.2% 2′, 7′-dichlorofluorecein in 95% ethanol or by 10% cupricsulfate in 8% phosphoric acid. The wax triester, cholesterol ester andtriglyceride spots were scraped, 1 ml of 5% HCl-methanol added andheated at 100° C. for 1 h (Miyazaki et al., 2000). The methyl esterswere analyzed by gas-liquid chromatography using cholesterolheptadecanoate, triheptadecanoate and heptadecanoic acid as internalstandard. Free cholesterol, cholesterol ester and triglycerides contentsof eyelid and plasma were determined by enzymatic assays (Sigma StLouis, Mo. and Wako Chemicals, Japan).

[0262] Isolation and Analysis of RNA

[0263] Total RNA was isolated from livers using the acidguanidinium-phenol-chloroform extraction method (Bernlohr et al., 1985).Twenty micrograms of total RNA was separated by 1.0% agarose/2.2 Mformaldehyde gel electrophoresis and transferred onto nylon membrane.The membrane was hybridized with ³²P-labeled SCD1 and SCD2 probes. pAL15probe was used as control for equal loading (Miyazaki, M., Kim, Y. C.,Gray-Keller, M. P., Attie, A. D., and Ntambi, J. M. (2000). Thebiosynthesis of hepatic cholesterol esters and triglycerides is impairedin mice with a disruption of the gene for stearoyl-CoA desaturase 1. JBiol Chem 275, 30132-8).

[0264] SCD Activity Assay

[0265] Stearoyl-CoA desaturase activity was measured in liver microsomesessentially as described by Shimomura et al. (Shimomura, I., Shimano,H., Korn, B. S., Bashmakov, Y., and Horton, J. D. (1998). Nuclear sterolregulatory element-binding proteins activate genes responsible for theentire program of unsaturated fatty acid biosynthesis in transgenicmouse liver. J Biol Chem 273, 35299-306.). Tissues were homogenized in10 vol. of buffer A (0.1M potassium buffer, pH 7.4). The microsomalmembrane fractions (100,000×g pellet) were isolated by sequentialcentrifugation. Reactions were performed at 37° C. for 5 min with 100 μgof protein homogenate and 60 μM of [1-¹⁴C]-stearoyl-CoA (60,000 dpm), 2mM of NADH, 0.1M of Tris/HCl buffer (pH 7.2). After the reaction, fattyacids were extracted and then methylated with 10% aceticchloride/methanol. Saturated fatty acid and monounsaturated fatty acidmethyl esters were separated by 10% AgNO₃-impregnated TLC usinghexane/diethyl ether (9:1) as developing solution. The plates weresprayed with 0.2% 2′, 7′-dichlorofluorescein in 95% ethanol and thelipids were identified under UV light. The fractions were scraped offthe plate, and the radioactivity was measured using a liquidscintillation counter. The enzyme activity was expressed as nmole min⁻¹mg⁻¹ protein.

[0266] Immunoblotting

[0267] Pooled liver membranes from 3 mice of each group were prepared asdescribed by Heinemann et al (Heinemann and Ozols, 1998). The sameamount of protein (25 μg) from each fraction was subjected to 10%SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-Ptransfer membranes at 4° C. After blocking with 10% non-fat milk in TBSbuffer (pH 8.0) plus Tween at 4° C. overnight, the membrane was washedand incubated with rabbit anti-rat SCD as primary antibody and goatanti-rabbit IgG-HRP conjugate as the secondary antibody. Visualizationof the SCD protein was performed with ECL western blot detection kit.

[0268] Histology

[0269] Tissues were fixed with neutral-buffered formalin, embedded inparaffin, sectioned and stained with hematoxylin and eosin.

[0270] This work was supported by a grant-in-aid from the American HeartAssociation-Wisconsin affiliate and in part by grant #DAMD17-99-9451from DOD.

[0271] Experimental Procedures for Asebia Mice:

[0272] Animals and Diets-Asebia homozygous (ab J/ab J or −/−) andheterozygous (+/ab J or +/−) mice were obtained from the JacksonLaboratory (Bar Harbor, Me.) and bred at the University of WisconsinAnimal Care Facility. In this study, comparisons are made between thehomozygous (−/−) and the heterozygous (+/−) mice since the latter areindistinguishable from normal mice. Mice were housed in a pathogen-freebarrier facility operating a 12-h light/12-h dark cycle. At 3 weeks ofage, these mice were fed ad libitum for 2 wks or 2 months, on laboratorychow diet or on a semi-purified diet containing 50% (% of total fattyacids) triolein or tripalmitolein. The semi-purified diet was purchasedfrom Harlan Teklad (Madison, Wis.) and contained: 18% vitamin freecasein, 5% soybean oil, 33.55% corn starch, 33.55% sucrose, 5%cellulose, 0.3%-L methionine, 0.1% choline chloride, salt mix (AIN-76A)and vitamin mix (AIN-76A). The fatty acid composition of theexperimental diets was determined by gas liquid chromatography. Thecontrol diet contained 11% palmitic acid (16:0), 23% oleic acid(18:1n-9), 53% linoleic acid (18:2n-6) and 8% linoleic acid (18:3n-3).The high triolein diet contained 7% 16:0, 50% 18:1n-9, 35% 18:2n-6 and5% 18:3n-3.

[0273] The high tripalmitolein diet contained 6% 16:0, 49% palmitoleicacid (16:1n-7), 12% 18:1 n-9, 27% 18:2n-6 and 4% 18:3n-3.

[0274] Animals were anesthetized at about 10:00 a.m. by intraperitonealinjection of pentobarbital sodium (0.08 mg/g of body weight) Nembutal,Abbot, North Chicago, Ill.). Liver was isolated immediately, weighed,and kept in liquid nitrogen. Blood samples were obtained from theabdominal vein.

[0275] Materials—Radioactive α-³²P]dCTP (3000 Ci/mmol) was obtained fromDupont Corp. (Wilmington, Del.). Thin layer chromatography plates (TLCSilica Gel G60) were from Merck (Darmstadt, Germany).[1-¹⁴C]-stearoyl-CoA, [³H]cholesterol and [1-¹⁴C]oleoyl-CoA werepurchased from American Radiolabeled Chemicals, Inc. (St Louis, Mo.).Immobilon-P transfer membranes were from Millipore (Danvers, Mass.). ECLWestern blot detection kit was from Amersham-Pharmacia Biotech, Inc.(Piscataway, N.J.). LT-1 transfection reagent was from PanVera (Madison,Wis.). All other chemicals were purchased from Sigma (St Louis, Mo.).The antibody for rat liver microsome SCD was provided by Dr. Juris Ozolsat University of Connecticut Health Center. pcDNA3-1 expression vectorSCD1 was provided by Dr. Trabis Knight at Iowa state university.

[0276] Lipid Analysis—Total lipids were extracted from liver and plasmaaccording to the method of Bligh and Dyer (Bligh, E. G., and Dyer, W. J.(1959) Can J Biochem Physiol 37, 911-917.), and phospholipids, freecholesterol, triglycerides and cholesterol esters were separated bysilica gel TLC. Petroleum ether/diethyl ether/acetic acid (80:30:1) wasused as a developing solvent. Spots were visualized by 0.2% 2′,7′-dichlorofluorecein in 95% ethanol or by 10% cupric sulfate in 8%phosphoric acid. The phospholipid, cholesterol ester and triglyceridespots were scraped, 1 ml of 5% HCl-methanol added and heated at 100 0 Cfor 1 h. The methyl esters were analyzed by gas-liquid chromatographyusing cholesterol heptadecanoate as internal standard (Lee, K. N.,Pariza, M. W., and Ntambi, J. M. (1998) Biochem. Biophys. Res. Commun.248, 817-821; Miyazaki, M., Huang, M. Z., Takemura, N., Watanabe, S.,and Okuyama, H. (1998) Lipids 33, 655-61). Free cholesterol, cholesterolester and triglycerides contents of liver and plasma were determined byenzymatic assays (Sigma St Louis, Mo. and Wako Chemicals, Japan).

[0277] Plasma Lipoprotein Analysis—Mice were fasted a minimum of 4 hoursand sacrificed by CO 2 asphyxiation and/or cervical dislocation. Bloodwas collected aseptically by direct cardiac puncture and centrifuged(13,000×g, 5 min, 4 0 C) to collect plasma. Lipoproteins werefractionated on a Superose 6HR 10/30 FPLC column (Pharmacia). Plasmasamples were diluted 1:1 with PBS, filtered (Cameo 3AS syringe filter,0.22 μm) and injected onto the column that had been equilibrated withPBS containing 1 mM EDTA and 0.02% NaN 3. The equivalent of 100 μl ofplasma was injected onto the column. The flow rate was set constant at0.3 ml/min. 500 μl fractions were collected and used for totaltriglyceride measurements (Sigma). Values reported are for totaltriglyceride mass per fraction. The identities of the lipoproteins havebeen confirmed by utilizing anti-ApoB immunoreactivity for LDL andAnti-Apo A1 immunoreactivity for HDL (not shown).

EXAMPLE 2 Demonstration of Significant Correlation Between the 18:1/18:0FFA Ratio and TG/HDL Levels in Humans

[0278] This example demonstrates, for the first time, that delta-9desaturase activity in humans correlates directly with serum levels oftriglyceride (VLDL) and inversely with serum HDL level and total serumcholesterol.

[0279] Experimental Design:

[0280] Plasma from a total of 97 individuals was analyzed for fatty acidcontent by gas chromatography (GC). Total free fatty acid (FFA) contentwas measured and the ratios of oleate to stearate (18:1/18:0) andpalmitoleate to palmitate (16:1/16:0) were computed, defined as thedesaturation indices, as above. We sought to find a relationship betweenthese ratios and three clinical indicators; plasma TG (triglyceride)levels, plasma HDL (high density lipoprotein) levels, and total plasmacholesterol.

[0281] Patient Sample:

[0282] The patient sample was chosen to maximize phenotypic diversity interms of HDL. Within our cohort, 21 individuals displayed a high HDLphenotype (>90^(th) percentile for age and sex), 12 individualsdisplayed a low HDL phenotype of unknown etiology (<5^(th) percentilefor age and sex), while six displayed a low HDL phenotype due tomutations in the ABCA1 gene. 33 individuals fall within normal HDLparameters (<90^(th) and >5^(th) percentile for age and sex).

[0283] We also attempted to diversity our sample in terms of TG levels,by including 9 individuals with Familial Combined Hyperlipidemia (FCHL)who have high TG and/or cholesterol as well as 16 additional controlindividuals with normal TG levels.

[0284] In some cases, multiple individuals from the same family weretested. Five of the six individuals with an ABCA1 mutation are part ofthe same family (NL-020). Multiple individuals were also tested fromother pedigrees segregating a low HDL phenotype that is not geneticallylinked to ABCA1. In this category, two affected individuals were testedfrom NL-008, while four affected individuals were tested from NL-001.The remaining six individuals with a low HDL phenotype are not relatedto one another, and were chosen from distinct pedigrees. Of thoseindividuals with high HDL, seven of them were unrelated to one another.It is not yet clear if the high HDL observed in these individuals has aclear genetic basis in family members. The remaining 14 individuals witha high HDL phenotype, six of them are from family HA-1 and eight arefrom a distinct family, HA-3. Unaffected individuals related to thosewith both low and high HDL were also tested.

[0285] Our cohort show wide variation in TG and HDL levels. In general,individuals with low HDL have high TG levels and those with high TGlevels tend to have low HDL levels. This relationship between TG and HDLhas been previously noted in the literature (Davis et al., 1980).

[0286] Analysis of fatty acid esters was determined as follows. Cellsfrom patient samples were washed twice with cold phosphate-bufferedsaline and total cellular lipids were extracted three times withCHCl₃/MeOH (2:1 v/v). The three lipid extractions were combined in ascrew-capped glass tube, dried under N2 gas at 40° C. in a heat block,and resuspended in toluene. Fatty acid methyl esters were produced fromBCI3/MeOH (Alltech, Deerfield Ill.), extracted with hexane, dried, andresuspended in hexane. Fatty acid methyl esters were identified using aHewlett-Packard 6890 gas chromatograph equipped with a 7683 autoinjector and an HP-5 column (30 m 0.25 mm, 0.25 μm film thickness)connected to a flame ionization detector set at 275° C. The injector wasmaintained at 250° C. The column temperature was held at 180° C. for 2min following injection, increased to 200° C. at 8° C./min, held at 200°C. for 15 min, and then increased to 250° C. at 8° C./min. Under theseconditions, the ?9?16:1?, 16:0?, ?9?18:1- and 18:0-methyl esters elutedat 9.2 min, 9.7 min, 15.3 min, and 16.4 min, respectively. See Lee etal. (1998). Biochem. Biophys. Res. Commun. 248:817-821; Miyazaki et al.(1998) Lipids 33:655-661; and Miyazaki M, Kim Y C, Gray-Keller M P,Attie A D, Ntambi J M. 2000. J. Biol. Chem. 275(39):30132-8.

[0287] Results

[0288] Linear regression analysis was carried out using the entire humandata set. The ratio of 18:1/18:0 showed a significant relationship to TGlevels (r²=0.39, p<0.0001) (FIG. 5a), as well as significantcorrelations to HDL levels (r²=0.12, p=0.0006) (FIG. 5b).

[0289] The 16:1/16:0 plasma fatty acid ratio was measured in a similarmanner, although the results were not as striking. A weak relationshipbetween the relative level of 16:1/16:0 to plasma TG levels was observed(r²=0.05, p=0.03) (FIG. 6), whereas the relationship between the16:1/16:0 ratio and HDL levels did not reach significance (not shown).In contrast to the 18:1/18:0 ratio, the 16:1/16:0 ratio did explain aportion of the variance in total cholesterol levels (r²=0.06,p=0.02)(not shown).

[0290] Overall the 18:1/18:0 ratio accounted for 18% of the variance intotal plasma fatty acid content (p=0.005) while the 16:1/16:0 ratioaccounted for 8% of the variation in this value (p=0.02), when theindividuals with FCHL and their associated controls were excluded fromthe analysis (not shown in the Figure).

[0291] Finally, for the portion of our sample for which Body Mass Index(BMI) values were available, we measured a positive correlation between18:1/18:0 ratios and BMI (r²=0.13, p=0.00) (data not shown).

[0292] The sample was stratified based on HDL levels to determine if therelationship between SCD activity (as measured by the 18:1/18:0 ratio)and TG levels was independent of the primary cause of the observeddyslipidemia.

[0293] A positive correlation was observed between 18:1/18:0 and TG inpersons with high HDL.

[0294] Analysis of those individuals with a high HDL phenotype (>90^(th)percentile) demonstrated a significant relationship between the18:1/18:0 ratio and TG levels (r²=0.40, p<0.005) (FIG. 7). Therelationship between 18:1/18:0 ratio and HDL levels in this group didnot reach significance (data not shown). The 16:1/16:0 ratio did notaccount for a significant proportion of the variance in totalcholesterol, TG or HDL levels in this subset of our cohort.

[0295] In order to determine if a stronger relationship between the18:1/18:0 index and TG levels would be apparent in a geneticallyhomogenous background, the HA-1 and HA-3 families were analyzedseparately. Both affected and unaffected family members were included inthe analysis. In both families, a similar relationship between 18:1/18:0and TG levels was observed (HA-1: r²=0.36, p=0.005 (FIG. 8a), HA-3:r²=0.32, p=0.009 (not shown in the figure)). The strength of theserelationships was similar to that observed in the entire cohort.18:1/18:0 ratios also correlated with HDL levels in HA-1, although thisrelationship did not reach significance in HA-3 (HA-1: r²=0.32, p=0.009(FIG. 8b), HA-3: r²=0.10, p=0.22 (not shown)).

[0296] A positive correlation was also observed between 18:1/18:0 and TGin those with low HDL. When all individuals with low HDL (<5^(th)percentile) were analyzed as a group, a significant relationship wasobserved between the 18:1/18:0 ratio and TG levels (r²=0.49, p=0.0009)(FIG. 9). As observed in our analysis of the high HDL patient subset,the relationship between the 18:1/18:0 ratio and HDL did not meetsignificance in the low HDL group (data not shown). In addition, nosignificant result was noted when the 16:1/16:0 ratio was regressed withHDL, TG and total cholesterol values.

[0297] Analysis of family NL-001, which segregated a low HDL phenotypeof unknown genetic etiology, and family NL-0020, which segregated anABCA1 mutation, tended towards the relationships noted above betweenfatty acid ratios and lipid parameters when affected individuals in eachfamily were considered. However, these results did not reach statisticalsignificance due to the small number of individuals analyzed in eachcase (NL-001: FIGS. 10a, b and NL0020: FIGS. 11a, b). A general trendtowards higher 18:1/18:0 ratios in older Tangiers patients was noted.This could be an effect independent of the disease, although an agedependent effect on the 18:1/18:0 ratio was noted in neither of the HA-1and HA-3 families nor in the entire cohort (not shown in FIG. 11).

[0298]FIG. 12 shows the relationship between the 18:1/18:0 ratio and TGlevels (r²=0.56, p=0.03) (FIG. 12a), HDL levels (r²=0.64, p=0.009) (FIG.12b) and total cholesterol levels (r²=0.50, p=0.03) in persons withFamilial Combined Hyperlipidemia (FCHL) (FIG. 12c).

[0299] Our analysis is the first demonstration in humans that SCDfunction, as measured by the 16:1/16:0 and 18:1118:0 desaturationindices, correlates positively with plasma TG levels and inversely withplasma HDL. Importantly, we observe this correlation irrespective of theunderlying cause of hyper- or hypo-triglyceridemia, suggesting that therelationship between SCD activity and TG levels is a generalized effect.Therefore, inhibition of SCD activity in humans is linked to decreasedserum TG (or VLDL) levels, increased total cholesterol levels, increasedHDL levels, and decreased body-mass-index (BMI), independent of theprimary cause of TG elevation. Importantly, SCD1 inhibitors could beused as a combination therapy in patients also being treated for FCHL.

[0300] In summary, when taken together, Examples 1 and 2 establish forthe first time a positive correlation between SCD1 activity and TGlevels in mammals, as well as an inverse correlation between SCD1activity and HDL in humans. Our analysis of the asebia and SCD1 KO aredefinitive in their implication of SCD1 as the major contributor to thedesaturation index. We have used this index as a surrogate for SCD1activity in our human studies. Thus, inhibitors of SCD1 function inmammals, including humans, are likely to both lower TG and raise HDL.

EXAMPLE 3 Plasma Fatty Acid Analysis in a Mouse Model of Dyslipidemia

[0301] In order to confirm the above described relationship observed inhumans between the 18:1/18:0 desaturation index and TG levels. We alsoperformed plasma fatty acid analysis in a mouse model of the humandisease FCHL. In the mouse hyperlipidemic strain (“hyplip”) TG levelsare elevated as compared to wild-type.

[0302] The hyperlipidemic mouse HcB-19 showed an elevated 18:1/18:0desaturation index. This mouse model of familial combined hyperlipidemia(HcB-19) displays elevated levels of TG, cholesterol, as well asincreased secretion of VLDL and apoB (Castellani et al, Mapping a genefor combined hyperlipidaemia in a mutant mouse strain. Nat Genet;18(4):374-7 (1998).

[0303] Plasma fatty acid analysis demonstrated that these animals have asignificantly elevated 18:1/18:0 ratio when compared to unaffectedcontrols of the parental strain (FIG. 13). The HcB-19 animals did not,however, show a significant elevation of the 16:1/16:0 index whencompared to controls. Therefore, we observe a positive correlationbetween the 18:1/18:0 desaturation index and TG levels in this animalmodel of FCHL.

EXAMPLE 4 Transcriptional Regulators of SCD1 and their use as DrugScreening Targets

[0304] This example reports, for the first time, the complete genomicpromoter sequence of human SCD1. This promoter is used herein toidentify regulatory elements that modulate and control SCD1 expressionin humans, and identifies regulatory proteins that are suitable targetsfor small molecule intervention to modulate expression of SCD1 inhumans.

[0305] The human SCD1 promoter sequence is set forth at SEQ ID No. 1.This sequence has not been accurately annotated in Genbank and has beenreported as 5′UTR in a number of records.

[0306]FIG. 14 illustrates the location of regulatory sequences andbinding sites in the homologous region of the mouse SCD1 and human SCD1promoter and 5′-flanking regions. The top scale denotes the positionrelative to the transcriptional start site. Important promoter sequenceelements are indicated.

[0307] The human SCD1 promoter structure is similar to that of the mouseSCD1 isoform and contains conserved regulatory sequences for the bindingof several transcription factors, including the sterol regulatoryelement binding protein (SREBP), CCAAT enhancer binding protein-alpha(C/EBPa) and nuclear factor-1 (NF-1) that have been shown totransactivate the transcription of the mouse SCD gene. Cholesterol andpolyunsaturated fatty acids (PUFAs) decreased the SCDpromoter-luciferase activity when transiently transfected into HepG2cells. The decrease in promoter activity in the reporter constructcorrelated with decreases in endogenous SCD mRNA and protein levels.Transient co-transfection into HepG2 cells of the human SCDpromoter-luciferase gene construct together with expression vector forSREBP revealed that SREBP trans-activates the human SCD promoter. Ourstudies indicate that like the mouse SCD1 gene, the human SCD gene isregulated by polyunsaturated fatty acids and cholesterol at the level ofgene transcription and that SREBP plays a role in the transcriptionalactivation of this gene.

[0308] Construction of the Chimeric Promoter Luciferase Plasmid

[0309] A human placenta genomic library in bacteria-phage I EMBL3 wasscreened with a 2.0 kb PstI insert of the mouse pC3 cDNA (Ntambi, J. M.,Buhrow, S. A., Kaestner, K. H., Christy, R. J., Sibley, E., Kelly, T. J.Jr., and M. D. Lane. 1988. Differentiation-induced gene expression in3T3-L1 preadipocytes: Characterization of a differentially expressedgene encoding stearoyl-CoA desaturase. J. Biol. Chem. 263: 17291-17300.) as a radioactive probe and seven plaques were isolated. Two of theseplaques were purified to homogeneity, the DNA isolated and designatedHSCD1 and HSCD3. A DNA primer based on the sequence corresponding to thefirst exon of the cDNA of the published human stearoyl-CoA desaturasegene (Zhang, L., G. E. Lan, S. Parimoo, K. Stenn and S. M. Proutey.1999. Human stearoyl-CoA desaturase: alternative transcripts generatedfrom a single gene by usage of tandem polyadenylation sites. Biochem. J.340: 255-264) was synthesized and used to sequence the two phage clonesby the dideoxy nucleotide chain termination method. A preliminarysequence was generated and primers upstream

[0310] 5′ NNNNGGTACCTTNNGAAAAGAACAGCGCCC 3′ SEQ ID No. 5

[0311] and downstream:

[0312] 5′ NNNNAGATCTGTGCGTGGAGGTCCCCG 3′ SEQ ID No. 6

[0313] were designed to amplify approximately 540 bases of the promoterregion upstream of the transcription start site: These primers containinserted restriction enzyme sites (underlined), Kpn1 for upstream, andBgIII for downstream, with a 4 base overhang region to allow restrictionenzyme digestion. PCR was then performed on the phage clones and theamplified 500 bp fragment was isolated from a 1% agarose gel.

[0314] The amplified fragment was digested with Kpn1 and BgIII and thencloned into the Kpn1 and BgIII sites of the pGL3 basic vector (Promega)that contains the luciferase reporter gene and transformed into DH5competent E. coli cells. Plasmid DNA was purified on Qiagen columns andsequenced by the dideoxynucleotide chain termination method using asprimers corresponding to DNA sequences within the multiple cloning sitebut flanking the inserted DNA. The SCD promoter luciferase geneconstruct that was generated was designated as pSCD-500.

[0315] Isolation and Analysis of RNA—Total RNA was isolated from HepG2cells using the acid guanidinium-phenol-chloroform extraction method.Twenty micrograms of total RNA was separated by 0.8% agarose/2.2 Mformaldehyde gel electrophoresis and transferred onto nylon membrane.The membrane was hybridized with ³²P-labeled human SCD cDNA probegenerated by PCR as follows: pAL15 probe was used as control for equalloading.

[0316] Immunoblotting—Cell extracts were prepared from HepG2 cells thathad been treated with the various fatty acids or cholesterol asdescribed by Heinemann et al (17). The same amount of protein (60 μg)from each fraction was subjected to 10% SDS-polyacrylamide gelelectrophoresis and transferred to Immobilon-P transfer membranes at 4°C. After blocking with 10% non-fat milk in TBS buffer (pH 8.0) plus 0.5%Tween at 4° C. overnight, the membrane was washed and incubated withrabbit anti-rat SCD as primary antibody (17) and goat anti-rabbitIgG-HRP conjugate as the secondary antibody. Visualization of the SCDprotein was performed with ECL western blot detection kit.

[0317] Effect of Cholesterol, Polyunsaturated Fatty Acids andArachidonic Acid on the Expression of hSCD1

[0318] Cell Culture and DNA transfections—HepG2 cells, were grown in LowGlucose DMEM supplemented with 10% Fetal Bovine Serum and 1%Penicillin/Streptomycin solution and maintained at 37 C, 5% CO₂ in ahumidified incubator. Cells were passaged into 6 cm dishes to give40-70% confluence in about 12-16 hours. Cells were then transfected with5 μg plasmid DNA per plate of pSCD-500 or the Basic PGL3 reporter aswell as well as the pRL-TK, internal controls (Promega) using the LT-1transfection reagent (Pan Vera). After 48 hours, cells were rinsed withPBS and then treated in Williams' E Media, a fatty acid-free media,containing insulin, dexamethasone, and appropriate concentrations ofalbumin-conjugated fatty acids as indicated in figures and legends.Cells were also treated with ethanol alone (as control) or cholesterol(10 μg/mL) and 25-OH cholesterol (1 μg/mL) dissolved in ethanol. Afteran additional 24 h, extracts were prepared and assayed for luciferaseactivity. Non-transfected cells were used as the blank and RenillaLuciferase was used as an internal control. Cell extracts were assayedfor protein according to Lowry, and all results were normalized toprotein concentration as well as to renilla luciferase counts. Eachexperiment was repeated at least three times, and all data are expressedas means±SEM.

[0319] Results:

[0320] The sequence of the amplified promoter region of the SCD1 gene isshown at SEQ ID. No. 1.

[0321] When compared to the mouse SCD1 promoter sequence, it was foundthat several functional regulatory sequences identified in the mouseSCD1 promoter are absolutely conserved at the nucleotide level and alsowith respect to their spacing within the proximal promoters of the twogenes (FIG. 14). Both the TTAATA homology, the C/EBPa and NF-1 are inthe same locations in both the mouse SCD1 and human promoters. Furtherupstream the sterol regulatory element (SRE) and the two CCAAT boxmotifs that are found in the polyunsaturated fatty acid responsiveelement (PUFA-RE) of the mouse SCD1 and SCD2 promoters. The spacing ofthese elements is conserved in the three promoters.

[0322] We tested whether the human SCD gene expression was alsorepressed by cholesterol and polyunsaturated fatty acids. Human HepG2cells were cultured and then treated with 100 μM arachidonic acid, DHAor 10 μg/ml cholesterol and 1 μg/ml of 25-hydroxycholesterol cholesterolas we have described previously. Total mRNA was isolated and subjectedto northern blot analysis using a probe corresponding to the human cDNAand generated by the PCR method using primers based on published humanSCD cDNA sequence. FIG. 15 shows that M, DHA and cholesterol decreasedthe human SCD mRNA expression in a dose dependent manner. The westernblot of the protein extracts of the cells treated with PUFAs andcholesterol shows that PUFAs and cholesterol decreased the levels of theSCD protein as well (data not shown).

[0323] To assess the possible effect of SREBP binding on the activity ofthe human SCD promoter the human luciferase promoter construct wasco-transfected in HepG2 cells together with an expression vectorcontaining SREBP1a. After 72 h, extracts of the transfected cells wereassayed for luciferase activity. Data were normalized to cell extractexpressing the Renilla luciferase as an internal control. As shown infigure SREBP transactivates the promoter in a dose dependent mannergiving rise to an increase up to 40-fold. This experiment shows thatSREBP plays a role in regulating the human SCD gene.

[0324] Published reports indicated that the mature form of SREBP, inaddition to activating the lipogenic genes, also mediates PUFA andcholesterol repression of lipogenic genes, including mouse SCD1. Toobserve the regulatory effects of mature SREBP-1a and PUFAs on theactivity of SCD promoters, HepG2 hepatic cells were transientlyco-transfected with 20 ng (per 6-cm dish) of plasmid DNA containing thehuman SCD promoter as described above but this time the transfectionswere carried out in the presence of cholesterol to inhibit thematuration of the endogenous SREBP and thus ensure that there was littlemature form of the endogenous SREBP present in the cells. Aftertransfection, the cells were then treated with, arachidonic acid, EPAand DHA as albumin complexes and luciferase activity was then assayedusing a luminometer. If SREBP mediates PUFA repression of the human SCDgene, SCD promoter activity would not diminish upon treatment thetransfected cells with PUFA. However addition of AA, EPA or DHAcontinued to repress SCD promoter activity with only a slightattenuation (data not shown). Thus, SREBP maturation does not seem toexhibit the selectivity required to explain PUFA control of SCD genetranscription suggesting that PUFA may utilize a different protein inaddition to the SREBP to repress human SCD gene transcription.

[0325] These results establish that hSCD1 is transcriptionally regulatedby SREBP, NF-Y, C/EBPalpha, PUFA-RE and alternate proteins andtranscription regulators. Each one of these proteins is therefore be anattractive drug screening target for identifying compounds whichmodulate SCD1 expression in a cell; and thereby being useful fortreating the human diseases, disorders and conditions which are taughtby the instant invention.

EXAMPLE 5 The SCD1 Knock-Out Displays Cutaneous and Ocular Abnormalities

[0326] To investigate the physiological functions of SCD, we havegenerated SCD1 knock-out (SCD1 −/−) mice. We found that the levels ofC16:1 were dramatically decreased in the tissues of SCD1 −/− micewhereas a dramatic decrease in C18:1 was noted only in liver where SCD1alone and not SCD2 is normally expressed. In tissues such as the eyelid,adipose and skin where both SCD1 and SCD2 are expressed, 18:1 was onlyslightly decreased. The monounsaturated fatty acids levels of the brainand eyeball which do not express SCD1 were unchanged. The liver and skinof the SCD−/− mice were deficient in cholesterol esters andtriglycerides while the eyelid in addition was deficient ineyelid-specific wax esters of long chain monounsaturated fatty acidsmainly C20:1. In addition the eyelid of the SCD−/− mice had higherlevels of free cholesterol. The SCD−/− mice exhibited cutaneousabnormalities with atrophic sebaceous gland and narrow eye fissure withatrophic meibomian glands which is similar to the dry eye syndrome inhumans. These results indicate that SCD1 deficiency can affect thesynthesis not only of monounsaturated fatty acids as components oftissue cholesterol ester and triglycerides but other lipids such as waxesters of the eyelid.

[0327] Gross Pathology and Histolgical Examination of SCD −/− Knock-OutMice.

[0328] SCD −/− mice were healthy and fertile but they have cutaneousabnormalities. These abnormalities started around weaning age (3-4weeks) with dry skin, fine epidermal scaling, and hair loss which becamemore severe with aging. In addition, the mice exhibited narrow eyefissures. Pathological examination of the skin and eyelids showed thewild-type mice had a prominent and well-differentiated sebaceous andmeibomian glands (data not shown). On the other hand, skin and eyelid ofSCD−/− appeared atrophic acinar cells in the sebaceous and meibomianglands (data not shown). No abnormalities were found in cornea andretina (data not shown).

[0329] SCD −/− Mice have Low Levels of Eyelid and Skin Neutral Lipids

[0330] We measured free cholesterol (FC) and cholesterol ester (CE),triglycerides and wax ester contents in the eyelid. Thin layerchromatography (TLC) of lipids extracted from eyelid of SCD1−/− micedemonstrated markedly reduced cholesterol ester and triglyceride and waxester levels compared to the lipids extracted from eyelid of wild-typemice (FIG. 16A). Table 3 compares eyelid lipid contents between SCD −/−and wild type mice. TABLE 3 Genotype +/+ −/− Cholesterol ester content(mg/g eyelid) 18.1 ± 0.7  4.8 ± 0.3 Free cholesterol content (mg/geyelid) 5.3 ± 0.5 8.4 ± 0.2 Wax triester ((mg/g eyelid) 36.8 ± 4.4  10.3± 0.8  Triglycerides (mg/g eyelid) 13.8 ± 0.6  5.5 ± 0.4

[0331] As shown in Table 3, and shows that the cholesterol ester contentin eyelid and skin of SCD1−/− mice was decreased by 74%, while freecholesterol increased by 1.75-fold. There was a reduction in the CE andtriglyceride level in the liver of the SCD−/− as well but there was nodifference in free cholesterol content in liver (data not shown). Thetriglyceride and wax ester contents in the eyelid of the SCD−/− micedecreased by 60% and 75%, respectively.

[0332]FIG. 16B shows use of a different solvent according to Nicolaidset al (Nicolaides, N., and Santos, E. C. (1985). The di- and triestersof the lipids of steer and human meibomian glands. Lipids 20, 454-67)consisting of hexane/benzene (45:65) was used to resolve the differentwax esters shows that the triester is the major wax ester. Thesetriesters as well as the diesters were decreased by 72% in the SCD−/−mice. The eyelid of wax triester content decreased by 72% in the SCD−/−mice. Similar to eyelid, cholesterol ester and triglyceride contents inthe skin of SCD−/− mice decreased by 43% and 53%, respectively whilefree cholesterol increased by 1.9-fold (Table 3, and FIG. 16A and B).Finally, the absolute monounsaturated fatty acid content in eachfraction was dramatically reduced in the SCD1−/− mice with correspondingincreases in the saturated fatty acids (data not shown).

[0333] Dietary 18:1 Did Not Restore Abnormalities of Skin and Eyelid inSCD −/− Mice

[0334] Oleate is one of the most abundant fatty acids in the diet. Thecellular monounsaturated fatty acids used for cholesterol ester andtriglyceride synthesis, could be synthesized either de novo by FattyAcid Synthase and SCD or by incorporation of exogenous oleate indirectlyfrom the diet. To determine whether dietary oleate could substitute forthe endogenously synthesized oleate and restore the hair, skin and eyeabnormalities of the SCD−/− mice, we supplemented the semi-purifiedmouse diets with high levels of 18:1n-9 (50% of total fat) as triolein,and then fed these diets to SCD−/− mice for 2 weeks. However, theseabnormalities were not restored by these diets which contained highmonounsaturated fatty acids. This suggests that SCD1 specific inhibitorswould act to reduce TG levels regardless of diet. Instead, cholesterolester, wax ester and triglyceride levels in the eyelids of SCD −/− micefed with high 18:1n-9 were still lower than those of SCD +/+ mice (datanot shown), suggesting that endogenously synthesized monounsaturatedfatty acids are required for the synthesis of the cholesterol esterstriglycerides and wax esters of mebum.

[0335] In the present study, we have established SCD1 null mice and haveshown that SCD deficiency caused substrate-selective andtissue-selective expression. The level of palmitoleate in SCD −/− miceis decreased by greater than 50% in all tissues including liver, whichexpressed SCD1 in wild-type mice. On the other hand, the alternations ofoleate level were tissue-specific.

[0336] Similar to asebia mice which have a spontaneous mutation of SCD1,SCD −/− mice exhibited abnormalities of hair growth, skin, and eye withcomplete penetrance. These phenotypes were noticeable from weaning age.Histological examination of the skin and eye lid showed that the atropicsebaceous gland in the skin and meibomian gland in the border of eyelidwhere SCD1 is abundantly expressed, lacked sebaceous and meibomiansecreted lipids, the so-called, sebum and mebum, respectively. In fact,we found that neutral lipids including triglyceride, several kinds ofwax esters and cholesterol ester which are known to be components ofsebum and mebum, were markedly reduced in the eyelid and also from theepidermis (data not shown) of the SCD −/− mice.

[0337] Chronic blepharitis similar to the eye lid abnormalities we havedescribed in the SCD −/− mice, is one of the most common frustratingdisease in humans. Shine and McCulley (Shine, W. E., and McCulley, J. P.(1998). Keratoconjunctivitis sicca associated with meibomian secretionpolar lipid abnormality. Arch Ophthalmol 116, 849-52) have reported thatchronic blephatitis may be due to lipid abnormalities in mebum. Thenature of these lipid abnormalities were not characterized in detail.They however, found that mebum from patients with meibomiankeratoconjunctivitis have decreased levels of oleic acid, a majorproduct of SCD whereas that from patients with meibomian seborrhea haveincreased levels of 18:1. These observations, together with our presentstudy, suggest that the alternation of SCD activity can be implicated inchronic blepharitis. Thus, the SCD may become a potential target for thedevelopment of therapeutic and preventive drugs for the treatment of eyediseases.

[0338] Promoter Sequence of Human Stearoyl-CoA Desaturase 1 ggtccccgccccttccagag agaaagctcc cgacgcggga tgccgggcag aggcccagcg SEQ ID No. 1gcgggtggaa gagaagctga gaaggagaaa cagaggggag ggggagcgag gagctggcggcagagggaac agcagattgc gccgagccaa tggcaacggc aggacgaggt ggcaccaaattcccttcggc caatgacgag ccggagttta cagaagcctc attagcattt ccccagaggcaggggcaggg gcagaggccg ggtggtgtgg tgtcggtgtc ggcagcatcc ccggcgccctgctgcggtcg ccgcgagcct cggcctctgt ctcctccccc tcccgccctt acctccacgcgggaccgccc gcgccagtca actcctcgca ctttgcccct gcttggcagc ggataaaagggggctgagga aataccggac acggtcaccc gttgccagct ctagccttta aattcccggctcggggacct ccacgcaccg cggctagcgc cgacaaccag ctagcgtgca aggcgccgcggctcagcgcg taccggcggg cttcgaaacc gcagtcctcc ggcgaccccg aactccgctccggagcctca gccccct

REFERENCE LIST

[0339] 1. Mihara, K. Structure and regulation of rat liver microsomalstearoyl-CoA desaturase gene. J. Biochem. (Tokyo) 108, 1022-1029 (1990).

[0340] 2. Thiede, M. A. & Strittmatter, P. The induction andcharacterization of rat liver stearyl-CoA desaturase mRNA. J. Biol.Chem. 260, 14459-14463 (1985).

[0341] 3. Kaestner, K. H., Ntambi, J. M., Kelly, T. J., Jr. & Lane, M.D. Differentiation-induced gene expression in 3T3-L1 preadipocytes. Asecond differentially expressed gene encoding stearoyl-CoA desaturase.J. Biol. Chem. 264, 14755-14761 (1989).

[0342] 4. Ntambi, J. M. et al. Differentiation-induced gene expressionin 3T3-L1 preadipocytes. Characterization of a differentially expressedgene encoding stearoyl-CoA desaturase. J. Biol. Chem. 263, 17291-17300(1988).

[0343] 5. Zhang, L., Ge, L., Parimoo, S., Stenn, K. & Prouty, S. M.Human stearoyl-CoA desaturase: alternative transcripts generated from asingle gene by usage of tandem polyadenylation sites. Biochem. J. 340(Pt 1), 255-264 (1999).

[0344] 6. Zheng, Y. et al. Scd1 is expressed in sebaceous glands and isdisrupted in the asebia mouse [letter]. Nat Genet. 23, 268-270 (1999).

[0345] 7. Sundberg, J. P. et al. Asebia-2J (Scd1(ab2J)): a new alleleand a model for scarring alopecia. Am. J. Pathol. 156, 2067-2075 (2000).

[0346] 8. Miyazaki, M., Kim, Y. C., Keller, M. P., Attie, A. D. &Ntambi, J. M. The biosynthesis of hepatic cholesterol esters andtriglycerides is impaired in mice with a disruption of the gene forstearoyl-CoA desaturase 1. J. Biol. Chem. (2000).

[0347] 9. Spector, A. A. & Yorek, M. A. Membrane lipid composition andcellular function. J. Lipid Res. 26, 1015-1035 (1985).

[0348] 10. Enser, M. & Roberts, J. L. The regulation of hepaticstearoyl-coenzyme A desaturase in obese-hyperglycaemic (ob/ob) mice byfood intake and the fatty acid composition of the diet. Biochem. J. 206,561-570 (1982).

[0349] 11. Enser, M. The role of insulin in the regulation of stearicacid desaturase activity in liver and adipose tissue fromobese—hyperglycaemic (ob/ob) and lean mice. Biochem. J. 180, 551-558(1979).

[0350] 12. Enser, M. Desaturation of stearic acid by liver and adiposetissue from obese-hyperglycaemic mice (ob/ob). Biochem. J. 148, 551-555(1975).

[0351] 13. Jones, B. H. et al. Adipose tissue stearoyl-CoA desaturasemRNA is increased by obesity and decreased by polyunsaturated fattyacids. Am. J. Physiol 271, E44-E49 (1996).

[0352] 14. Kim, Y. C., Gomez, F. E., Fox, B. G. & Ntambi, J. M.Differential regulation of the stearoyl-CoA desaturase genes bythiazolidinediones in 3T3-L1 adipocytes. J. Lipid Res. 41, 1310-1316(2000).

[0353] 15. Li, J. et al. Partial characterization of a cDNA for humanstearoyl-CoA desaturase and changes in its mRNA expression in somenormal and malignant tissues. Int. J. Cancer 57, 348-352 (1994).

[0354] 16. Wood, C. B. et al. Reduction in the stearic to oleic acidratio in human malignant liver neoplasms. Eur. J. Surg. Oncol. 11,347-348 (1985).

[0355] 17. Habib, N. A. et al. Stearic acid and carcinogenesis. Br. J.Cancer 56, 455-458 (1987).

[0356] 18. Tronstad, K. J., Berge, K., Bjerkvig, R., Flatmark, T. &Berge, R. K. Metabolic effects of 3-thia fatty acid in cancer cells.Adv. Exp. Med. Biol. 466, 201-204 (1999).

[0357] 19. DeWille, J. W. & Farmer, S. J. Postnatal dietary fatinfluences mRNAS involved in myelination. Dev. Neurosci. 14, 61-68(1992).

[0358] 20. Garbay, B. et al. Regulation of oleoyl-CoA synthesis in theperipheral nervous system: demonstration of a link with myelinsynthesis. J. Neurochem. 71, 1719-1726 (1998).

[0359] 21. Marcelo, C. L., Duell, E. A., Rhodes, L. M. & Dunham, W. R.In vitro model of essential fatty acid deficiency. J. Invest Dermatol.99, 703-708 (1992).

[0360] 22. Tebbey, P. W. & Buttke, T. M. Stearoyl-CoA desaturase geneexpression in lymphocytes [published erratum appears in Biochem BiophysRes Commun Sep. 16, 1992;187(2):1201]. Biochem. Biophys. Res. Commun.186, 531-536 (1992).

[0361] 23. Tebbey, P. W. & Buttke, T. M. Molecular basis for theimmunosuppressive action of stearic acid on T cells [published erratumappears in Immunology October 1990;71(2):306]. Immunology 70, 379-386(1990).

[0362] 24. Stampfer et al. A prospective study of cholesterol,apolipoproteins, and the risk of myocardial infraction. N. Engl. J. Med.325, 373-381 (1991).

[0363] 25. Schmidt et al. Clustering of dyslipidemia, hyperuricemia,diabetes, and hypertension and its association with fasting insulin andcentral and overall obesity in a general population. AtherosclerosisRisk in Communities Study Investigators Metabolism 45 (6):699-706(1996).

[0364] 26. Park et al. Inhibition of hepatic stearoyl-CoA desaturaseactivity by trans-10, cis-12 conjugated linoleic acid and itsderivatives. Biochim Biophys Acta. 1486(2-3):285-92 (2000).

[0365] 27. Choi et al. The trans-10,cis-12 isomer of conjugated linoleicacid downregulates stearoyl-CoA desaturase 1 gene expression in 3T3-L1adipocytes. J Nutr. 130(8):1920-4 (2000)

1 6 1 617 DNA Homo sapiens 1 ggtccccgcc ccttccagag agaaagctcc cgacgcgggatgccgggcag aggcccagcg 60 gcgggtggaa gagaagctga gaaggagaaa cagaggggagggggagcgag gagctggcgg 120 cagagggaac agcagattgc gccgagccaa tggcaacggcaggacgaggt ggcaccaaat 180 tcccttcggc caatgacgag ccggagttta cagaagcctcattagcattt ccccagaggc 240 aggggcaggg gcagaggccg ggtggtgtgg tgtcggtgtcggcagcatcc ccggcgccct 300 gctgcggtcg ccgcgagcct cggcctctgt ctcctccccctcccgccctt acctccacgc 360 gggaccgccc gcgccagtca actcctcgca ctttgcccctgcttggcagc ggataaaagg 420 gggctgagga aataccggac acggtcaccc gttgccagctctagccttta aattcccggc 480 tcggggacct ccacgcaccg cggctagcgc cgacaaccagctagcgtgca aggcgccgcg 540 gctcagcgcg taccggcggg cttcgaaacc gcagtcctccggcgaccccg aactccgctc 600 cggagcctca gccccct 617 2 24 DNA ArtificialSequence Description of Artificial Sequence PCR Primer for Exon 6. 2gggtgagcat ggtgctcagt ccct 24 3 20 DNA Artificial Sequence Descriptionof Artificial Sequence PCR Primer for the neo gene. 3 atagcaggcatgctggggat 20 4 28 DNA Artificial Sequence Description of ArtificialSequence PCR amplification primer. 4 cacaccatat ctgtccccga caaatgtc 28 530 DNA Artificial Sequence Description of Artificial Sequence UpstreamPCR amplification primer. 5 nnnnggtacc ttnngaaaag aacagcgccc 30 6 27 DNAArtificial Sequence Description of Artificial Sequence Downstream PCRamplification primer. 6 nnnnagatct gtgcgtggag gtccccg 27

What is claimed is:
 1. A method for identifying, from a library of testcompounds, a therapeutic agent which is useful in humans for thetreatment of a disorder or condition relating to serum levels oftriglyceride or VLDL comprising a) providing a screening assay havingSCD1 biological activity; b) contacting said screening assay with a testcompound; and c) subsequently measuring said biological activity;wherein a test compound which modulates said biological activity is saidtherapeutic agent, or an analog thereof.
 2. A screening assay foridentifying compounds useful in human for the treatment of a disorder orcondition relating to serum levels of triglyceride, or VLDL, saidscreening assay comprising a) a screening assay having SCD1 biologicalactivity; wherein a test compound which modulates said SCD1 biologicalactivity in said screening assay is a compound, or an analog thereof,which is useful for said treatment.
 3. A method for identifying, from alibrary of test compounds, a therapeutic agent which is useful in humansfor the treatment of a disorder or condition relating to serum levels oftriglyceride or VLDL, comprising a) an assay having measurable SCD1biological activity; wherein a test compound that modulates SCD1biological activity upon contact with said assay is said therapeuticagent or an analog thereof.
 4. The assay of claim 1-3 wherein saidcompound is an antagonist or inhibitor of SCD1 biological activity. 5.The assay of claim 1-3 wherein said compound is an agonist of SCD1biological activity
 6. The assay of claims 1-5 wherein inhibitor doesnot substantially inhibit the biological activity in a human of adelta-5 desaturase, delta-6 desaturase or fatty acid synthetase
 7. Theassay of claims 1-5 further comprising the step of assaying saidtherapeutic agent to further select compounds which do not substantiallyinhibit in a human the activity of delta-5 desaturase, delta-6desaturase or fatty acid synthetase.
 8. The screening assay of claim1-3, wherein SCD1 biological activity is measured by an assay selectedfrom among: a) SCD1 polypeptide binding affinity; b) SCD1 desaturaseactivity in microsomes; c) SCD1 desaturase activity in a whole cellassay d) quantification of SCD1 gene expression level; and e)quantification of SCD1 protein level.
 9. A cell line containing arecombinant SCD1 protein.
 10. A cell line containing the recombinantSCD1 protein of claim 9 which is used in a screening assay foridentifying compounds that inhibit SCD1 biological activity and areuseful for treatment in a human of a disorder or condition relating toserum levels of triglyceride or VLDL
 11. An assay employing the cellline of claim 9 wherein the identified compound is further selected fromamong those compounds that do not substantially inhibit in humans thebiological activity of delta-5 desaturase, delta-6 desaturase or fattyacid synthetase.
 12. A recombinant cell line comprising the SCD1promoter nucleic acid sequence of SEQ ID No. 1 operably linked to areporter gene construct.
 13. Use of the recombinant cell line of claim12 in a screening assay for identifying compounds which are useful forthe treatment in humans of a disorder or condition relating to serumlevels of triglyceride or VLDL.
 14. An isolated stearoyl-CoA desaturase(SCD) nucleic acid encoded by the polynucleotide sequence comprising SEQID No. [SCD1 cDNA]
 15. A reporter gene construct comprising the SCD1promoter nucleic acid sequence of SEQ ID No. 1 operably linked to areporter gene.
 16. A vector comprising the nucleic acid of claim 14 or15.
 17. An isolated stearoyl-CoA desaturase protein.
 18. A method foridentifying a compound which binds to or interacts with the polypeptideof claim 17 comprising: a) contacting the polypeptide of claim 17 or acell expressing the polypeptide of claim 17 with a test compound; and b)determining whether the polypeptide binds to or interacts with the testcompound.
 19. The method of claim 18 wherein the binding of the testcompound to the polypeptide is detected by a method selected from thegroup consisting of: a) detection of binding by direct detection of testcompound/polypeptide binding; b) detection of binding using acompetition binding assay; and c) detection of binding using an assayfor SCD1 biological activity.
 20. A method for modulating the activityof the polypeptide of claim 17 comprising contacting the polypeptide ora cell expressing the polypeptide with a compound which binds to thepolypeptide in sufficient amount to modulate the activity of thepolypeptide.
 21. A screening assay employing SCD1 nucleic acid of claim14 and/or SCD1 polypeptide of claim 17 for use in identifying compoundsuseful for treatment of a disorder or condition relating to serum levelsof triglyceride or VLDL.
 22. A method of treating a disease or conditionin a human selected from among a disorder or condition relating to serumlevels of triglyceride or VLDL, said method consisting essentially ofinhibition of the activity of SCD1 protein in said human.
 23. The methodof claim 22, wherein said inhibitor does not substantially inhibitactivity of delta-5 desaturase, delta-6 desaturase or fatty acidsynthetase.
 24. Use of a compound for treatment of a disorder orcondition relating to serum levels of triglyceride or VLDL, wherein saidcompound or analog thereof was identified by its ability to modulateSCD1 biological activity in an assay of claim 1-3.
 25. Use of a compoundin a human for treatment of a disorder or condition relating to serumlevels of triglyceride or VLDL, wherein said use of said compound oranalog thereof was first identified by said compound's ability tomodulate SCD1 biological activity in an assay of claim 1-3.
 26. Amodulator of SCD1 biological activity which is useful in humans fortreatment of a disorder or condition relating to serum levels oftriglyceride or VLDL, identified by a screening assay wherein saidmodulator detectably modulates SCD1 biological activity.
 27. Acomposition which is useful in humans for treatment of a disorder orcondition relating to serum levels of triglyceride or VLDL, firstidentified by a screening assay wherein said composition modulates thebiological activity of SCD1.
 28. A method for identifying, from alibrary of test compounds, a therapeutic agent which is useful in humansfor the treatment of a disorder or condition relating to serum levels oftriglyceride, VLDL, HDL, LDL, total cholesterol, reverse cholesteroltransport or production of secretions from mucous membranes,monounsaturated fatty acids, wax esters, and the like, comprising a)providing a screening assay having a measurable biological activity of avertebrate delta-9 stearoyl-CoA desaturase; b) contacting said screeningassay with a test compound; and c) subsequently measuring saidbiological activity; wherein a test compound which modulates saidbiological activity is said therapeutic agent, or an analog thereof. 29.Use of a compound in humans for treatment of a disorder or conditionrelating to serum levels of triglyceride, VLDL, HDL, LDL, totalcholesterol, reverse cholesterol transport or production of secretionsfrom mucous membranes, monounsaturated fatty acids, wax esters, and thelike, wherein said compound or analog thereof was identified by itsability to modulate a measurable biological activity of a vertebratedelta-9 stearoyl-CoA desaturase in an assay of claim
 28. 30. Use of acompound in humans for treatment of a disorder or condition relating toserum levels of triglyceride, VLDL, HDL, LDL, total cholesterol, reversecholesterol transport or production of secretions from mucous membranes,monounsaturated fatty acids, wax esters, and the like, wherein said useof said compound or analog thereof was first identified by saidcompound's ability to modulate a measurable biological activity of avertebrate delta-9 stearoyl-CoA desaturase in an assay of claim
 28. 31.The method of claims 28-30 wherein the compound does not in humanssubstantially inhibit fatty acid synthetase, delta-5 desaturase ordelta-6 desaturase.
 32. A method for up-regulating the ABCA1 gene in anindividual comprising the step of administering to that individual anagent which lowers the level of activity of stearoyl-CoA desaturase(SCD1) protein in that individual.
 33. A method as claimed in claim 32wherein the agent is an inhibitor which inhibits the enzymatic activityof the SCD1 protein.
 34. A method as claimed in claim 33 wherein theinhibitor is selected from the group consisting of a thia-fatty acid, aconjugated linoleic acid, and a cyclopropenoid fatty acid.
 35. A methodas claimed in claim 34 wherein the thia-fatty acid is selected from thegroup consisting of a 9-thiastearic acid and a fatty acid having asulfoxy moiety.
 36. A method as claimed in claim 34 wherein theconjugated linoleic acid is a trans-10, cis 12 conjugated linoleic acid.37. A method as claimed in claim 34 wherein the cyclopropenoid fattyacid is selected from the group consisting of sterulic acid and malvalicacid.
 38. A method as claimed in claim 32 wherein the agent inhibits theSCD1 protein by inhibiting the transcription of an SCD1 gene.
 39. Amethod as claimed in claim 38 wherein the inhibitor is selected from thegroup consisting of a thiazoladinedione compound and a polyunsaturatedfatty acid.
 40. A method as claimed in claim 39 wherein thethiazoladinedione compound is selected from the group consisting ofBRL49653, Pioglitazone, Ciglitazone, Englitazone, and Troglitazone. 41.A method as claimed in claim 39 wherein the polyunsaturated fatty acidis selected from the group consisting of dodecahexaenoic acid,arachidonic acid, and linoleic acid.
 42. A method as claimed in claim 33wherein the inhibitor inhibits the SCD1 protein by inhibiting a proteinselected from the group consisting of a cytochrome b₅ protein, aNADH-cytochrome b₅ reductase protein, and a terminal cyanide-sensitivedesaturase protein.
 43. A method for elevating high density lipoprotein(HDL) particles in an individual comprising the step of administering tothat individual an inhibitor of an SCD1 protein.
 44. A method forreducing very low density lipoprotein (VLDL) particles in an individualcomprising the step of administering to that individual an inhibitor ofan SCD1 protein.
 45. A method for reducing plasma triglyceridesparticles in an individual comprising the step of administering to thatindividual an inhibitor of an SCD1 protein.
 46. A method for enhancingthe cellular efflux of phospholipids and/or cholesterol in an individualcomprising the step of administering to that individual an inhibitor ofan SCD1 protein.
 47. A method for inhibiting atherosclerosis in anindividual comprising the step of administering to that individual aninhibitor of an SCD1 protein.
 48. A method for treating diabetes andinsulin resistance in an individual comprising the step of administeringto that individual an inhibitor of an SCD1 protein expression oractivity.
 49. A method of testing compounds for their effects on ABCA1activity comprising the steps of feeding an amount of the test compoundsto s subject and monitoring the effect on the level of SCD1 activity inthe subject.
 50. A method for identifying, from a library of testcompounds, a therapeutic agent which is useful in humans for thetreatment of a disorder or condition relating to serum levels of HDL,LDL, total cholesterol, reverse cholesterol transport or production ofsecretions from mucous membranes, monounsaturated fatty acids, waxesters, and the like, comprising a) providing a screening assay havingSCD1 biological activity; b) contacting said screening assay with a testcompound; and c) subsequently measuring said biological activity;wherein a test compound which modulates said biological activity is saidtherapeutic agent, or an analog thereof.
 51. A screening assay foridentifying compounds useful in human for the treatment of a disorder orcondition relating to serum levels of HDL, LDL, total cholesterol,reverse cholesterol transport or production of secretions from mucousmembranes, monounsaturated fatty acids, wax esters, and the like, saidscreening assay comprising a) a screening assay having SCD1 biologicalactivity; wherein a test compound which modulates said SCD1 biologicalactivity in said screening assay is a compound, or an analog thereof,which is useful for said treatment.
 52. A method for identifying, from alibrary of test compounds, a therapeutic agent which is useful in humansfor the treatment of a disorder or condition relating to serum levels ofHDL, LDL, total cholesterol, reverse cholesterol transport or productionof secretions from mucous membranes, monounsaturated fatty acids, waxesters, and the like, comprising a) an assay having measurable SCD1biological activity; wherein a test compound that modulates SCD1biological activity upon contact with said assay is said therapeuticagent or an analog thereof.
 53. The assay of claim 1-3 wherein saidcompound is an antagonist or inhibitor of SCD1 biological activity. 54.The assay of claim 1-3 wherein said compound is an agonist of SCD1biological activity
 55. The assay of claims 1-5 wherein inhibitor doesnot substantially inhibit the biological activity in a human of adelta-5 desaturase, delta-6 desaturase or fatty acid synthetase
 56. Theassay of claims 1-5 further comprising the step of assaying saidtherapeutic agent to further select compounds which do not substantiallyinhibit in a human the activity of delta-5 desaturase, delta-6desaturase or fatty acid synthetase.
 57. The screening assay of claim1-3, wherein SCD1 biological activity is measured by an assay selectedfrom among: a) SCD1 polypeptide binding affinity; b) SCD1 desaturaseactivity in microsomes; c) SCD1 desaturase activity in a whole cellassay d) quantification of SCD1 gene expression level; and e)quantification of SCD1 protein level.
 58. A cell line containing arecombinant SCD1 protein.
 59. A cell line containing the recombinantSCD1 protein of claim 9 which is used in a screening assay foridentifying compounds that inhibit SCD1 biological activity and areuseful for treatment in a human of a disorder or condition relating toserum levels of HDL, LDL, total cholesterol, reverse cholesteroltransport or production of secretions from mucous membranes,monounsaturated fatty acids, wax esters, and the like.
 60. An assayemploying the cell line of claim 9 wherein the identified compound isfurther selected from among those compounds that do not substantiallyinhibit in humans the biological activity of delta-5 desaturase, delta-6desaturase or fatty acid synthetase.
 61. A recombinant cell linecomprising the SCD1 promoter nucleic acid sequence of SEQ ID No. 1operably linked to a reporter gene construct.
 62. Use of the recombinantcell line of claim 12 in a screening assay for identifying compoundswhich are useful for the treatment in humans of a disorder or conditionrelating to serum levels of HDL, LDL, total cholesterol, reversecholesterol transport or production of secretions from mucous membranes,monounsaturated fatty acids, wax esters, and the like.
 63. An isolatedstearoyl-CoA desaturase nucleic acid encoded by the polynucleotidesequence comprising SEQ ID NO.
 1. 64. A reporter gene constructcomprising the SCD1 promoter nucleic acid sequence of SEQ ID No. 1operably linked to a reporter gene.
 65. A vector comprising the nucleicacid of claim 14 or
 15. 66. An isolated stearoyl-CoA desaturase protein.67. A method for identifying a compound which binds to or interacts withthe polypeptide of claim 17 comprising: a) contacting the polypeptide ofclaim 17 or a cell expressing the polypeptide of claim 17 with a testcompound; and b) determining whether the polypeptide binds to orinteracts with the test compound.
 68. The method of claim 18 wherein thebinding of the test compound to the polypeptide is detected by a methodselected from the group consisting of: a) detection of binding by directdetection of test compound/polypeptide binding; b) detection of bindingusing a competition binding assay; and c) detection of binding using anassay for SCD1 biological activity.
 69. A method for modulating theactivity of the polypeptide of claim 17 comprising contacting thepolypeptide or a cell expressing the polypeptide with a compound whichbinds to the polypeptide in sufficient amount to modulate the activityof the polypeptide.
 70. A screening assay employing SCD1 nucleic acid ofclaim 14 and/or SCD1 polypeptide of claim 17 for use in identifyingcompounds useful for treatment of a disorder or condition relating toserum levels of HDL, LDL, total cholesterol, reverse cholesteroltransport or production of secretions from mucous membranes,monounsaturated fatty acids, wax esters, and the like.
 71. A method oftreating a disease or condition in a human selected from among adisorder or condition relating to serum levels of HDL, LDL, totalcholesterol, reverse cholesterol transport or production of secretionsfrom mucous membranes, monounsaturated fatty acids, wax esters, and thelike, said method consisting essentially of inhibition of the activityof SCD1 protein in said human.
 72. The method of claim 22, wherein saidinhibitor does not substantially inhibit activity of delta-5 desaturase,delta-6 desaturase or fatty acid synthetase.
 73. Use of a compound fortreatment of a disorder or condition relating to serum levels of HDL,LDL, total cholesterol, reverse cholesterol transport or production ofsecretions from mucous membranes, monounsaturated fatty acids, waxesters, and the like, wherein said compound or analog thereof wasidentified by its ability to modulate SCD1 biological activity in anassay of claim 1-3.
 74. Use of a compound in a human for treatment of adisorder or condition relating to serum levels of HDL, LDL, totalcholesterol, reverse cholesterol transport or production of secretionsfrom mucous membranes, monounsaturated fatty acids, wax esters, and thelike, wherein said use of said compound or analog thereof was firstidentified by said compound's ability to modulate SCD1 biologicalactivity in an assay of claim 1-3.
 75. A modulator of SCD1 biologicalactivity which is useful in humans for treatment of a disorder orcondition relating to serum levels of HDL, LDL, total cholesterol,reverse cholesterol transport or production of secretions from mucousmembranes, monounsaturated fatty acids, wax esters, and the like,identified by a screening assay wherein said modulator detectablymodulates SCD1 biological activity.
 76. A composition which is useful inhumans for treatment of a disorder or condition relating to serum levelsof HDL, LDL, total cholesterol, reverse cholesterol transport orproduction of secretions from mucous membranes, monounsaturated fattyacids, wax esters, and the like, first identified by a screening assaywherein said composition modulates the biological activity of SCD1. 77.A process for identifying a SCD1-modulating agent, comprising: a)contacting under physiological conditions a chemical agent and amolecule having or inducing SCD1 activity; b) detecting a change in theactivity of said molecule having or inducing SCD1 activity followingsaid contacting; thereby identifying an SCD1 modulating agent.
 78. Theprocess of claim 77 wherein said molecule having or inducing SCD1activity is a polypeptide having such activity.
 79. The process of claim77 wherein said molecule having or inducing SCD1 activity is apolynucleotide encoding a polypeptide having such activity.
 80. Theprocess of claim 77 wherein said molecule having or inducing SCD1activity is a polypeptide modulating the activity of a polynucleotideencoding a polypeptide having such activity.
 81. The process of claim 77wherein said change in activity is an increase in activity.
 82. Theprocess of claim 77 wherein said change in activity is a decrease inactivity.
 83. The process of claim 77 wherein said contacting isaccomplished in vivo.
 84. The process of claim 83 wherein saidcontacting in step (a) is accomplished by administering said chemicalagent to an animal afflicted with a triglyceride (TG)- or very lowdensity lipoprotein (VLDL)-related disorder and subsequently detecting achange in plasma triglyceride level in said animal thereby identifying atherapeutic agent useful in treating a triglyceride (TG)- or very lowdensity lipoprotein (VLDL)-related disorder.
 85. The process of claim 84wherein said animal is a human.
 86. The process of claim 84 wherein saidchange in SCD1 activity in said animal is a decrease in activity. 87.The process of claim 84 or 85 wherein said SCD1 modulating agent doesnot substantially inhibit the biological activity of a delta-5desaturase, delta-6 desaturase or fatty acid synthetase.
 88. The processof claim 77 or 84 wherein said detectable change in SCD1 activity isdetected by detecting a change in: a) SCD1 polypeptide binding affinity;b) SCD1 desaturase activity in microsomes; c) SCD1 desaturase activityin a whole cell; d) SCD1 gene expression; or e) SCD1 protein level. 89 Arecombinant cell line comprising a recombinant SCD1 protein.
 90. Theprocess of claim 88 wherein said whole cell of (c) is derived from thecell line of claim
 89. 91. The process of claim 90 wherein said SCD1modulating agent does not substantially inhibit in humans the biologicalactivity of delta-5 desaturase, delta-6 desaturase or fatty acidsynthetase.
 92. A recombinant cell line comprising the SCD1 promoternucleic acid sequence of SEQ ID No. 1 operably linked to a reporter geneconstruct.
 93. The process of claim 88 wherein said whole cell of (c) isderived from the cell line of claim
 92. 94. An isolated stearoyl-CoAdesaturase encoded by the polynucleotide sequence comprising SEQ ID No.[SCD1 cDNA]
 95. A reporter gene construct comprising the SCD1 promoternucleic acid sequence of SEQ ID No. 1 operably linked to a reportergene.
 96. A vector comprising the nucleic acid of claim 94 or
 95. 97. Anisolated polypeptide having stearoyl-CoA reductase activity.
 98. Aprocess for identifying a chemical agent that binds to or interacts withthe polypeptide of claim 97 comprising: a) contacting the polypeptide ofclaim 97 or a cell expressing the polypeptide of claim 97 with achemical agent; and b) detecting binding or interaction of the chemicalagent with said polypeptide.
 99. The process of claim 98 wherein thebinding of the chemical agent to the polypeptide is detected by a methodselected from the group consisting of: a) direct detection of chemicalagent/polypeptide binding; b) detection of binding by competitionbinding assay; and c) detection of binding by assay for SCD1 biologicalactivity.
 100. A process for modulating the activity of the polypeptideof claim 97 comprising contacting the polypeptide or a cell expressingthe polypeptide with a compound that binds to the polypeptide insufficient amount to modulate the activity of the polypeptide.
 102. Theprocess of claim 77 or 84 wherein said molecule having or inducing SCD1activity is selected from the group consisting of the SCD1 nucleic acidof claim 94 and/or SCD1 polypeptide of claim
 97. 103. A process fortreating a human afflicted with a disease or condition relating to serumlevels of triglyceride or VLDL comprising inhibiting SCD1 activity insaid human.
 104. The method of claim 100 wherein said inhibition of SCD1activity is not accompanied by substantial inhibition of activity ofdelta-5 desaturase, delta-6 desaturase or fatty acid synthetase.
 105. Aprocess for treating a human patient afflicted with a disorder orcondition relating to serum levels of triglyceride or VLDL comprisingadministering to said patient a therapeutically effective amount of anagent whose therapeutic activity was first identified by the process ofclaim 77 or
 84. 106. A modulator of SCD1 activity which is useful inhumans for treatment of a disorder or condition relating to serum levelsof triglyceride or VLDL wherein said activity was first identified byits ability to modulate SCD1 activity.
 107. A process for identifying avertebrate delta-9 stearoyl-CoA desaturase-modulating agent, comprising:a) contacting under physiological conditions a chemical agent and amolecule having or inducing vertebrate delta-9 stearoyl-CoA desaturaseactivity; b) detecting a change in the activity of said molecule havingor inducing vertebrate delta-9 stearoyl-CoA desaturase activityfollowing said contacting;  thereby identifying a vertebrate delta-9stearoyl-CoA desaturase modulating agent.
 109. The process of claim 106wherein said contacting in step (a) is accomplished by administeringsaid chemical agent to an animal afflicted with a disorder or conditionrelated to serum levels of triglyceride, VLDL, HDL, LDL, totalcholesterol, reverse cholesterol transport or production or secretion ofmucous membranes, monounsaturated fatty acids, wax esters, and likeparameters, detecting a change in the activity of said molecule havingor inducing vertebrate delta-9 stearoyl-CoA desaturase activityfollowing said contacting and thereby identifying a therapeutic agentuseful in treating a triglyceride, VLDL, HDL, LDL, total cholesterol, orproduction or secretion of mucous membranes, monounsaturated fattyacids, wax esters, and like disease-related disorder.
 109. A process fortreating a human patient afflicted with a disease or condition relatingto serum levels of triglyceride, VLDL, HDL, LDL, total cholesterol,reverse cholesterol transport or production or secretion of mucousmembranes, monounsaturated fatty acids, wax esters, and like parameters,comprising administering to said human patient a therapeuticallyeffective amount of an agent for which such therapeutic activity wasidentified by the process of claim
 106. 110. The process of claim107-109 wherein the modulating agent does not substantially inhibitfatty acid synthetase, delta-5 desaturase or delta-6 desaturase ofhumans.
 111. A process for identifying, from a library of testcompounds, a therapeutic agent which is useful in humans for thetreatment of a disorder or condition relating to serum levels oftriglyceride or very low density lipoprotein (VLDL) comprising a)providing a screening assay having stearoyl-Coenzyme A desaturase type 1(SCD1) biological activity as a component thereof; b) contacting saidSCD1 activity with a test compound; c) administering to a human acompound found to modulate said activity in (b); and (d) detecting achange in serum level of triglyceride or VLDL in said human followingsaid administering; thereby identifying an agent useful in the treatmentof a disorder or condition relating to serum levels of triglyceride orvery low density lipoprotein (VLDL).
 112. The process of claim 111wherein said agent is an antagonist or inhibitor of SCD1 biologicalactivity.
 113. The process of claim 111 wherein said agent is an agonistof SCD1 biological activity.
 114. The process of claim 112 wherein saidinhibitor does not substantially inhibit the biological activity in ahuman of a delta-5 desaturase, delta-6 desaturase or fatty acidsynthetase 115 The process of claim 111 further comprising the step ofassaying said therapeutic agent to further select compounds which do notsubstantially inhibit in a human the activity of delta-5 desaturase,delta-6 desaturase or fatty acid synthetase.
 116. The process of claim111 wherein said SCD1 biological activity is measured by an assayselected from among: a) SCD1 polypeptide binding affinity; b) SCD1desaturase activity in microsomes; c) SCD1 desaturase activity in awhole cell assay d) quantification of SCD1 gene expression level; and e)quantification of SCD1 protein level.
 117. The process of claim 116employing the cell of claim
 9. 118. The process of claim 117 wherein theidentified compound is further selected from among those compounds thatdo not substantially inhibit in humans the biological activity ofdelta-5 desaturase, delta-6 desaturase or fatty acid synthetase. 119.The process of claim 116 employing SCD1 nucleic acid of claim 14 and/orSCD1 polypeptide of claim 17 for use in identifying compounds useful fortreatment of a disorder or condition relating to serum levels oftriglyceride or VLDL.